NOR gene compositions and methods for use thereof

ABSTRACT

The current invention provides nucleic acid sequences encoding the NOR gene. Compositions comprising this sequence are described, as are plants transformed with such compositions. Further provided are methods for the expression of the NOR gene. The methods of the invention include the direct creation of transgenic plants with the NOR gene by genetic transformation, as well as by plant breeding methods. The sequences of the invention represent a valuable new tool for the creation of transgenic plants, preferably having one or more added beneficial characteristics.

This application claims the priority of U.S. Provisional ApplicationSer. No. 60/143,357, filed Jul. 12, 1999, the disclosure of which isspecifically incorporated herein by reference in its entirety.

The government may own rights in this invention subject to grant numbersUSDA-NRICGP 92-373000-7653, USDA-NRICGP 92-373000-1575, Texas AdvancedTechnology Program 999902037, and USDA-NRICGP 91-373000-6418.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the NOR gene. Morespecifically, it relates to methods and compositions for themodification of plant phenotypes with the NOR gene.

2. Description of the Related Art

The ripe phenotype is the summation of biochemical and physiologicalchanges occurring at the terminal stage of fruit development renderingthe organ edible and desirable to seed dispersing animals and valuableas an agricultural commodity. These changes, although variable amongspecies, generally include modification of cell wall ultrastructure andtexture, conversion of starch to sugars, increased susceptibility topost-harvest pathogens, alterations in pigmentbiosynthesis/accumulation, and heightened levels of flavor and aromaticvolatiles (Rhodes, 1980; Hobson and Grierson, 1993). Several of thesesripening attributes translate to decreased shelf-life and high inputharvest, shipping and storage practices, particularly via changes infirmness and the overall decrease in resistance to microbial infectionof ripe fruit. Currently acceptable techniques for minimizing theconsequences of undesirable ripening characteristics include prematureharvest, controlled atmosphere storage, pesticide application, andchemically induced ripening to synchronize the timing of maturation.Unfortunately, added production, shipping and processing expenses, inaddition to reduced fruit quality, are often the consequence of thesepractices, challenging both the competitiveness and long termsustainability of current levels of crop production.

Although most fruit display modifications in color, texture, flavor, andpathogen susceptibility during maturation, two major classifications ofripening fruit, climacteric and non-climacteric, have been utilized todistinguish fruit on the basis of respiration and ethylene biosynthesisrates. Climacteric fruit such as tomato, cucurbits, avocado, banana,peaches, plums, and apples, are distinguished from non-climactericfruits such as strawberry, grape and citrus, by their increasedrespiration and ethylene biosynthesis rates during ripening (Grierson,1986). Ethylene has been shown to be necessary for the coordination andcompletion of ripening in climacteric fruit via analysis of inhibitorsof ethylene biosynthesis and perception (Yang, 1985; Tucker and Brady,1987), in transgenic plants blocked in ethylene biosynthesis (Klee etal., 1991; Oeller et al., 1991; Picton et al., 1993 a), and throughexamination of the Never-ripe (Nr) ethylene perception mutant of tomato(Lanahan et al., 1994).

Considerable attention has been directed toward elucidating themolecular basis of ripening in the model system of tomato during recentyears (reviewed in Spiers and Brady, 1991; Gray et al., 1992 and 1994;Giovannoni, 1993; Theologis 1992 and Theologis et al., 1993). Thecritical role of ethylene in coordinating climacteric ripening at themolecular level was first observed via analysis of ethylene inducibleripening-related gene expression (Tucker and Laties, 1984; Lincoln etal., 1987; Maunders et al., 1987; DellaPenna et al., 1989; Starrett andLaties; 1993). Several ripening genes, including ACC synthase and ACCoxidase, have been shown via antisense gene repression to have profoundinfluences on the onset and degree of ripening (Hamilton et al., 1990;Oeller et al., 1991). Although the sum effect of this research has beena wealth of information pertaining to the regulation of ethylenebiosynthesis and its role in ripening, the molecular basis ofdevelopmental cues which initiate ripening-related ethylenebiosynthesis, and additional aspects of ripening not directly influencedby ethylene, remain largely unknown (Theologis et al., 1993).

Single locus mutations which attenuate or arrest the normal ripeningprocess, and do not ripen in response to exogenous ethylene, have beenidentified in tomato and are likely to represent lesions in regulatorycomponents necessary for initiation of the ripening cascade, includingethylene biosynthesis (Tigchelaar et al., 1978; Grierson, 1987;Giovannoni, 1993; Hobson and Grierson, 1993; Gray et al., 1994). Onesuch mutation, the Nr mutation, has been identified and represents agene responsible for ethylene perception and/or signal transduction andis a tomato homologue of the Arabidopsis Ethylene response 1 (Etr1) gene(Yen et al., 1995; Wilkinson et al., 1995).

Tomato has served as a model for ripening of climacteric fruit.Ripening-related genes have been isolated via differential geneexpression patterns (Slater et al., 1985, Lincoln et al., 1987, Pear etal., 1989, Picton et al., 1993b) and biochemical function (DellaPenna etal., 1986; Sheehy et al., 1987; Ray et al., 1988; Biggs and Handa, 1989;Harriman and Handa, 1991; Oeller et al., 1991; Yelle et al., 1991).Promoter analysis of ripening genes has been performed via examinationof promoter/reporter construct activities in transient assay systems andtransgenic plants. The result has been the identification of cis-actingpromoter elements which are responsible for both ethylene andnon-ethylene regulated aspects of ripening (Deikman et al., 1992;Montgomery et al., 1993). Trans-acting factors which interact with thesepromoters also have been identified via gel-shift and footprintexperiments, although none have been isolated or cloned (Deikman andFischer, 1988; Cordes et al., 1989; Montgomery et al., 1993).

The in vivo functions of several ripening-related genes includingpolygalacturonase, pectinmethylesterase, ACC synthase, ACC oxidase, andphytoene synthase have been tested via antisense gene repression and/ormutant complementation in transgenic tomatoes. For example, the cellwall pectinase, polygalacturonase, was shown to be necessary forripening-related pectin depolymerization and pathogen susceptibility,however, the inhibition of PG expression had minimal effects on fruitsoftening (Smith et al., 1988, Giovannoni et al., 1989, Kramer et al.,1990). Significant reduction in rates of ethylene evolution resulting ininhibition of most ripening characteristics was observed in both ACCsynthase and ACC oxidase antisense mutants (Oeller et al., 1991;Hamilton et al., 1990). Non-ripening antisense fruit were subsequentlyrestored to normal ripening phenotype with the application of exogenousethylene.

Further analysis of transgenic tomatoes inhibited in ethylenebiosynthesis demonstrates that climacteric ripening represents acombination of both ethylene mediated and developmental control(Theologis et al., 1993). Although antisense ACC synthase tomatoes whichfailed to produce ethylene did not ripen, gene expression analysisdemonstrated that several ripening-related genes, includingpolygalacturonase and E8 are expressed in the absence of ethylene. Thisobservation confirms the presence of a developmental (or non-ethyleneregulated) component of ripening. In fact, an ethylene requirement wasobserved for translation but not transcription of polygalacturonasemRNA, suggesting interaction between ethylene and non-ethylenecomponents of ripening for expression of at least a subset of ripeninggenes (Theologis et al., 1993).

While the above studies have provided some insight into the ripeningprocess in plants, there is still a great need in the art for novelmethods and compositions for the creation of plants having enhancedphenotypes. In particular, there is a need in the art for the isolationthe RIN and NOR genes. The isolation of these genes would allow thecreation of novel transgenic plants altered in their fruitcharacteristics and/or ethylene responsiveness, and having one or moreadded beneficial properties.

SUMMARY OF THE INVENTION

In one aspect, the current invention provides an isolated nucleic acidsequence comprising the NOR gene. In one embodiment of the invention,the NOR gene may be further defined as isolatable from the nucleic acidsequence of SEQ ID NO:1, SEQ ID NO:6 or SEQ ID NO:7. In particularembodiments of the invention, the invention provides an isolated nucleicacid corresponding to an open reading frame of the NOR cDNA, forexample, which may be denoted by the nucleotides as indicated by boldletters in FIG. 6.

In another aspect, the invention provides an isolated nucleic acidsequence having from about 17 to about 1209, about 25 to about 1209,about 30 to about 1209, about 40 to about 1209, about 60 to about 1209,about 100 to about 1209, about 200 to about 1209, about 400 to about1209, about 600 to about 1209, about 800 to about 1209, or about 1000 toabout 1209 contiguous nucleotides of the nucleic acid sequence of SEQ IDNO:6 or SEQ ID NO:7. Similarly, the invention provides such nucleic acidsegments from SEQ ID NO:1. In particular embodiments of the invention,the nucleic acid sequences of SEQ ID NO:6 and SEQ ID NO:7 are provided.In particular embodiments of the invention, a nucleic acid sequence ofthe invention may further comprising an enhancer, such as an intron. Anucleic acid sequence of the invention may also include atranscriptional terminator. Such sequences may be native to the NOR geneor heterologous from potentially any species.

In yet another aspect, the invention provides an expression vectorcomprising a NOR gene. Such a NOR gene may be in accordance with any ofthe NOR-containing sequences described herein. The expression vector maycomprise the NOR gene operably linked to a native or heterologouspromoter, either in sense or antisense orientation relative to thepromoter. Potentially any heterologous promoter may be used, forexample, a promoter is selected from the group consisting of CaMV ³⁵S,CaMV 19S, nos, Adh, actin, histone, ribulose bisphosphate carboxylase,R-allele, root cell promoter, α-tubulin, ABA-inducible promoter,turgor-inducible promoter, rbcS, corn sucrose synthetase 1, corn alcoholdehydrogenase 1, corn light harvesting complex, corn heat shock protein,pea small subunit RuBP carboxylase, Ti plasmid mannopine synthase, Tiplasmid nopaline synthase, petunia chalcone isomerase, bean glycine richprotein 1, CaMV 35s transcript, Potato patatin, actin, cab, PEPCase andS-E9 small subunit RuBP carboxylase promoter. In still furtherembodiments of the invention, the expression vector may comprise anyselectable marker, for example, a selectable marker selected from thegroup consisting of phosphinothricin acetyltransferase, glyphosateresistant EPSPS, aminoglycoside phosphotransferase, hygromycinphosphotransferase, neomycin phosphotransferase, dalapon dehalogenase,bromoxynil resistant nitrilase, anthranilate synthase and glyphosateoxidoreductase.

The expression vector may be either circular, for example, as in thecase of a plasmid vector, or could be a linear nucleic acid segment,such as an expression cassette isolated from a plasmid. In particularembodiments of the invention, the vector is a plasmid vector. Theexpression vector may further comprise other elements, such as a nucleicacid sequence encoding a transit peptide, or potentially any terminator,for example, a heterologous terminator such as the nos terminator.

In still yet another aspect, the invention provides a transgenic plantcomprising a stably transformed expression vector, such as thosedescribed above. The transgenic plant may be any type of plant, and inparticular embodiments of the invention is a tomato plant. In furtherembodiments of the invention, the transgenic plant may be a fertile R₀transgenic plant. Also included in the invention is a seed of such afertile R₀ transgenic plant, wherein said seed comprises said expressionvector. The transgenic plant may be a progeny plant of any generation ofa fertile R₀ transgenic plant, wherein said R₀ transgenic plantcomprises said expression vector. The invention also includes a seed ofsuch a progeny plant, wherein said seed comprises said expressionvector.

In still yet another aspect, the invention provides a crossed fertiletransgenic plant prepared according to the method comprising the stepsof: (i) obtaining a fertile transgenic plant comprising a selected DNAcomprising a NOR gene; (ii) crossing said fertile transgenic plant withitself or with a second plant lacking said selected DNA to prepare theseed of a crossed fertile transgenic plant, wherein said seed comprisessaid selected DNA; and (iii) planting said seed to obtain a crossedfertile transgenic plant. In one embodiment of the invention, a seed isprovided of such a crossed fertile transgenic plant, wherein said seedcomprises said selected DNA. The crossed fertile transgenic plant may beof any species, for example, a tomato plant. The plant may also beinbred or hybrid.

In still yet another aspect, the invention provides a method ofmanipulating the phenotype of a plant comprising the steps of: (i)obtaining an expression vector comprising a NOR gene in sense orantisense orientation; (ii) transforming a recipient plant cell withsaid expression vector; and (iii) regenerating a transgenic plant fromsaid recipient plant cell, wherein the phenotype of said plant isaltered based on the expression of said NOR gene in sense or antisenseorientation. Any method of transforming a plant may be used inaccordance with the invention, including, microprojectile bombardment,PEG mediated transformation of protoplasts, electroporation, siliconcarbide fiber mediated transformation, or Agrobacterium-mediatedtransformation. In particular embodiments of the invention,Agrobacterium-mediated transformation is used and the plant is a tomatoplant.

In still yet another aspect, the invention provides a method of plantbreeding comprising the steps of: (i) obtaining a transgenic plantcomprising a selected DNA comprising a NOR gene; and (ii) crossing saidtransgenic plant with itself or a second plant. The plant may be of anyspecies and may be inbred or hybrid. In particular embodiments of theinvention, this method further comprises the steps of: (iii) collectingseeds resulting from said crossing; (iv) growing said seeds to produceprogeny plants; (v) identifying a progeny plant comprising said selectedDNA; and (vi) crossing said progeny plant with itself or a third plant.In one embodiment of the invention, the second plant and third plant areof the same genotype. The second and third plants may also be inbredplants.

In still yet another aspect, the invention provides a transgenic plantcell stably transformed with a selected DNA comprising a NOR gene. Thecell may be from any plant species, for example, a cell from a tomatoplant. The selected may comprise any of the NOR gene comprising nucleicacid compositions disclosed herein, for example, the expression vectorcompositions described herein above. Such compositions include the openreading frame of the NOR gene, as provided in SEQ ID NO:6 or SEQ ID NO:7and demarcated in FIG. 6.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein. The file of this patent containsat least one drawing executed in color. Copies of this patent with colordrawing(s) will be provided by the Patent and Trademark Office uponrequest and payment of the necessary fee.

FIG. 1. T-DNA constructs for delivery of sense or antisense NOR genecDNA (CD-11) sequences into plant genomes. The base plasmid, termedNOR-pBI121 Sense/Antisense (Kanamicin resistant—NPTII), had anapproximate size of 13.0 kb. Abbreviations are as follows: FB, LB: rightand left T-DNA borders, respectively; Nos-pro: nopaline synthasepromoter driving expression of the NPTII gene; NPTII: neomycinphosphotransferase (kanamycin resistance) gene; nos-ter: transactiontermination sequence from the nopaline synthase gene; HindIII, SphI,PstI, Xbal, BamHI, SmaI, EcoRI, SpeI, SacI: DNA restrictionendonucleases (enzymes); and Sma/EcoRV: the resulting chimeric sequenceis recognized by neither enzyme.

FIG. 2. Manipulation of fruit ripening and carotenoid accumulation withthe tomato NOR gene. Shown are representative control and transformedfruit from tomato a line of the genotype nor/nor in the cultivar MH1 andtransformed with NOR-pBI121 Sense (FIG. 1). Primary transformants (T0)were confirmed for transgene integration via DNA gel-blot analysis andsubsequently self-pollinated. Resulting seed were harvested and grown(T1 generation) and analyzed for transgene segregation. Representativefully mature fruit from T1 nor/nor individuals that either harbor thesense NOR transgene (+) or have segregated it out (−) are shown. Insummary, transgene expression in the mutant background partiallyrecovers the non-ripening phenotype and confers ripening. In thisparticular line, relatively low expression of the transgene was observedas compared to expression of NOR in normally ripening (Nor/Nor) fruit.Representative normal (Nor/Nor) and nearly isogenic mutant (nor/nor)cultivar MH1 tomato fruit are shown as controls. The partial recovery ofripening in the nor/nor fruit harboring the NOR-pBI121 (+) transgeneverified the isolation of the NOR gene. Furthermore, the partialripening phenotype observed in this line demonstrated that regulatedexpression of the NOR gene can be used to create a range of degrees ofripening and ripening-associated characteristics (e.g., carotenoidaccumulation, ripe flavor, nutrient composition, softness, pathogensusceptibility).

FIG. 3. DNA agarose gel showing the Nor versus nor alleles as PCRamplification products. Genomic DNA was isolated from normal (N/N) andhomozygous nor mutant (n/n) nearly isogenic control lines (cultivarMH1), in addition to individuals from a Nor/Nor X Nor/nor back-cross(BC) population. DNA was amplified with one PCR primer common to thecoding region of both the Nor and nor alleles and separately with eitherone primer specific to the Nor or nor alleles, respectively. Theallele-specific primers were based on the 2 bp deletion whichdistinguished the normal (Nor) versus mutant (nor) allele (see FIG. 5).PCR reactions with the normal (Nor) allele primer were loaded on the topportion of the gel, and those employing the mutant (nor) allele primerwere loaded on the bottom portion of the gel. PCR reactions from thesame individual plant but amplified separately with each allele-specificprimer were loaded directly above and below each other to facilitatescoring. The normal (N) and mutant (n) alleles are indicated above eachlane and represent the corresponding genotype as determined by analysisof band amplification.

FIG. 4. Expression of the NOR gene through plant development and innormal and mutant fruit. RNA gel-blot analysis of expression using theNOR full-length cDNA as probe. Expression was induced in the transitionfrom mature green to breaker fruit but was not detected in anyadditional tissues examined including combined cotyledons and hypocotyls(C/H), leaves, senescing leaves (S/Leaf), stems or roots. It was notedthat expression was also reduced in identically aged nor (nor/nor)mutant fruit.

FIG. 5. DNA sequence of the region of tomato chromosome 10 harboring theNOR gene (SEQ ID NO:7). The sense genomic DNA sequence of the completetranscribed region is shown in the 5′-3′ orientation. The codingsequence is in upper case while non-translated sequences including thetwo NOR gene introns are in lower case.

FIG. 6. Corrected DNA sequence of the NOR full-length cDNA (CD-11) (SEQID NO:6). The full cDNA sequence is shown in 5′-3′ orientation. Lowercase letters refer to non-translated portions of the transcript whilethe upper case letters refer to the translated (coding) sequence.

DETAILED DESCRIPTION OF THE INVENTION

The ripening of fleshy fruits represents a system of eukaryoticdevelopment unique to plants as well as an important component ofagricultural quality and productivity. Greater understanding of thegenetic and molecular basis of the ripening process will promote bothour collective understanding of plant development and yield tools usefulfor sustaining and improving agricultural productivity and quality,while minimizing impact on the resources necessary for production. Thecurrent invention provides such understanding by providing the nucleicacids encoding the NOR gene. By providing these sequences, the inventionprovides, for the first time, the ability to use genetic transformationtechniques to manipulate a variety of plant characteristics which areassociated with these genes in ways that cannot be accomplished viatraditional breeding strategies including direct DNA transfer to speciesother than tomato.

In tomato, ripening occurs over a period of several days, depending onvariety, and is characterized by softening, pectin solubilization,increased respiration and ethylene biosynthesis, enhanced pathogensusceptibility, heightened palatability, and accumulation of thecharacteristic red and orange carotenoid pigments lycopene andbeta-carotene, respectively. NOR and also the fruit ripening gene RINsegregate as single traits, result in nearly complete inhibition ofnormal ripening as defined above, and their effects on ripening cannotbe restored via application of exogenous ethylene (Tigchelaar et al.,1978). The ripening phenotypes displayed by RIN and NOR demonstrate thatthe gene products encoded by the normal alleles at these loci areinvolved in the primary regulation of ripening (Hobson and Grierson,1993; Giovannoni 1993; Gray et al., 1994). Because virtually nothing isknown of the expression patterns or biochemical nature of the normal NORgene product, the inventors initiated a genetic map-based cloningstrategy for isolation of the corresponding normal allele. All of theprerequisite tools for implementation of this strategy are available intomato including 1) the mutations themselves, 2) DNA markers tightlylinked to both rin and nor, 3) large populations (>300 F2 progeny)segregating for target loci, 4) a library of high molecular weighttomato genomic DNA, and 5) gene transfer technology for verification ofcloned target genes via complementation of the recessive phenotype withthe dominant allele.

Tomato has served for decades as a model system for both plant geneticsand fruit ripening, in part resulting in the availability of the toolsfor ripening gene isolation mentioned above. Numerous mutationsregulating various aspects of tomato fruit ripening have been identifiedover the years, most of which result in alteration of pigmentbiosynthesis and/or accumulation without significant effects onadditional ripening characteristics (Rick, 1980; Grierson, 1986; Gray etal., 1994). Examples include the greenflesh (gf; Ramirez and Tomes,1964) and yellowflesh (r; Darby, 1978) mutants which inhibitripening-related chlorophyll degradation and lycopene accumulation,respectively. Tomato mutations exerting complete or nearly completeinhibition of overall ripening (i.e. blocking changes not just in colorbut also texture, ethylene biosynthesis, pathogen susceptibility, flavorand aroma) are few, the most extreme being rin and nor.

Neither mutation exerts any observable influence on aspects of plantdevelopment or morphology other than ripening, suggesting regulatoryroles limited primarily to fruit development (the rin mutation isassociated with the mc or macrocalyx phenotype; however, geneticevidence indicates that the rin mutant is actually a double mutant atthe linked RIN and MC loci Robinson and Tomes, 1968)). Fruit homozygousfor either rin or nor are similar in phenotype in that they attain fullsize, produce viable seed, yet remain firm and green for weeks afternormal fruit ripen. In addition, homozygous rin and nor mutant fruitfail to display climacteric respiration and ethylene biosynthesischaracteristic of normally ripening tomatoes (Tigchelaar et al., 1978),are highly resistant to microbial infection (Grierson, 1986), and areinhibited in their expression of ripening-related genes (DellaPenna etal., 1989; Picton et al., 1993). Although often referred to as recessivemutations, both rin and nor heterozygotes show significant effects onsome ripening parameters, including reduced pathogen susceptibility andsoftening, resulting in extended shelf-life (Tigchelaar et al., 1978;Biggs and Handa, 1989). For this reason, heterozygosity at the RIN locusin particular has seen increased commercial application in fresh markethybrids. Isolation of the RIN and NOR genes by the current inventorspermits optimization of controlled ripening via controlled expression intomato and potentially other fruit crop species. From a broaderstandpoint, the cloned RIN and NOR genes serve as cornerstones fromwhich to build a model system for analysis of the developmentalregulation of fruit ripening control.

I. Rationale and Significance of the Invention

Ripening is a unique and important plant process whose understanding hasgreat significance in the agricultural arts. Isolation of genesregulating both the ethylene and non-ethylene mediated components offruit ripening represents an important step in understanding the geneticbasis of this complex developmental pathway. Although most researchemphasis in recent years has been focused on elucidating thebiosynthesis and function of ethylene during climacteric fruit ripening,the genetic and molecular basis of the developmental regulators whichinitiate ripening ethylene biosynthesis, and control the non-ethylenemediated ripening pathway, had remained a mystery. The mutant phenotypesof the targeted nor locus demonstrates that this gene is essential fornormal ripening to occur and is a developmental regulator both ofethylene biosynthesis and non-ethylene regulated aspects of ripening. Inaddition, this gene may be related to those involved in the regulationof other developmental programs. Insights gained into the ripeningprocess as a result of the current invention will not only aid ourunderstanding of overall plant development, but may enhanceunderstanding of developmental processes in other eukaryotes as well.

From the standpoint of agriculture, ripening confers both positive andnegative attributes to the resulting commodity. While ripening impartsdesirable flavor, color, and texture, considerable expense and crop lossresult as a consequence of negative ripening characteristics. Forexample, ripening related increases in fruit pathogen susceptibility isa major contributor to fruit loss both before and after harvest. Thisgenetically regulated change in fruit physiology currently necessitatesthe use of pesticides, post-harvest fumigants, and controlled atmospherestorage and shipping mechanisms in attempts to minimize loss. Inaddition to being wasteful of energy and potentially harmful to theenvironment, such practices represent major expenses in fruitproduction.

The current inventors, however, have isolated the ripening regulatorygene NOR, which allows for the first time the genetic enhancementthrough manipulation of genes of positive ripening attributes andreduction of undesirable qualities in tomato and additional species. Theability to improve fruit quality while reducing energy use, productioncosts, and environmental impact will promote the long term productivityand sustainability of commercial agriculture.

The current inventors employed a map-based cloning approach for theisolation of the normal NOR locus in tomato. Tomato is the bestavailable system for the map-based cloning of ripening genes because ofthe availability of: 1) single locus mutations inhibiting normal fruitripening, 2) a high density RFLP map, 3) large populations segregatingfor targeted ripening loci, 4) a YAC library, and 5) establishedprocedures for transformation and regeneration. Also, gene products havenot been identified for either of the target genes, thus precludingimmunological cloning strategies. In addition, previous to the inventionone could only speculate concerning patterns of normal NOR geneexpression, thus exacerbating the already difficult task of identifyingappropriate stages for differential screening strategies. Therefore, thecurrent invention represents a major advance over the prior art,potentially allowing for the first time the creation of transgenicplants having greatly enhanced agronomic characteristics.

II. Alteration of Plant Phenotypes with NOR Nucleic Acid Compositions

(i) NOR Gene Function

The effects and thus potential uses of the NOR (non-ripening) gene canbe deduced from analysis of the well characterized mutation at the norlocus (see Tigchellar et al., 1978 and Giovannoni, 1993 for review).Further, the inventors have shown that the NOR gene mutation (nor)greatly inhibits the ripening process with minimal effects on otherplant tissues or even fruit prior to the onset of ripening.Consequently, the use of the NOR gene may be indicated for manipulationof fruit ripening. It is also apparent not only from the mutantphenotypes but also from the transgenic expression of the NOR gene(i.e., expression is primarily restricted to the tissues in whicheffects are observed, fruits) that normal effects are centered on thedeveloping flower, specifically, the carpels (fruit). Nevertheless,manipulation of NOR gene in non-fruit tissues via the tools ofbiotechnology could be expected to yield various potentially usefuleffects in non-fruit tissues as well (see examples below). It should benoted that subsequent reference to “normal” and “mutant” refers to thegenotypes Nor/Nor and nor/nor, respectively.

Fruit ripening is a complex process ultimately rendering the fruitpalatable and/or susceptible to biotic or abiotic process which resultin seed liberation and dispersal. While specific ripening attributesvary among species, the following general process are common to manyfruits (see Seymour et al., 1993 for review), including tomato, and areall have been shown to be influenced by the NOR gene viacharacterization of the corresponding mutant:

A) Degradation of the photosynthetic pigment chlorophyll andaccumulation of various pigment compounds (often carotenoids andflavonoids) resulting in changes of both color and nutritionalcomposition (Tigchellar et al., 1978; Yen et al., 1997).

B) Changes in cell wall metabolism and architecture resulting in effectson texture and susceptibility to pathogen infection with additionalimpacts on specific aspects of processing qualities including viscosityand texture of whole and chopped/pureed products (Tigchellar et al.,1978).

C) Changes in carbohydrate metabolism including the conversion of starchto simple sugars (Seymour et al., 1993).

D) Changes in aroma and production of associated volatile compounds.

E) Changes in ethylene hormone biosynthesis and perception (DellaPennaet al., 1989) which directly influence many of the specific ripeningattributes mentioned here but may also impact these and other areas viamechanisms not described above. Such processes include effects onpathogen susceptibility, senescence, abscission, seed germination,flowering, sex determination in cucurbits and general stress responses(temperature, drought, mechanical damage) See Ables et al., 1992 forreview.

Previous observations, some of which are referenced above, confirm thefunction of the NOR gene in most aspects of fruit ripening and suggestthat additional aspects of plant growth, development and response to theenvironment could be altered via expression of this gene in other planttissues via alternate promoters. As such, alteration of any of theforgoing phenotypes, as well as other phenotypes conferred by the NORgene, as well as plants altered in such ways, specifically form a partof the instant invention.

(ii) Examples of NOR Gene Use.

The NOR gene compositions provided by the inventors may find numeroususes in manipulation of plant phenotypes. Exemplary uses for the NORgene are described herein below, although those of skill in the art willrecognize that the examples are in no way limiting.

1. Control of Fruit Ripening and Quality

Though currently less widely used than the tomato rin(ripening-inhibitor) mutation, the nor mutation is currently used intomato breeding for development of hybrid lines withslow-ripening/long-shelf-life characteristics. The NOR gene couldsimilarly be used for manipulation and control of ripening withpotential for accelerated ripening of important early season crops,controlled or delayed ripening of crops permitting longer shippinghandling, storage and post-retail shelf-life. The fact that theinventors have provided the cloned NOR gene will permit its utilizationin species other than tomato. Specific examples of use would be inaccelerated ripening of early season melons for favorable marketposition and pricing, and ripening control of bananas andstrawberry—fruits which typically have short shelf-lives making shippingand handling more costly.

Next, modified expression of the NOR gene in ripening fruits may finduse in elevating levels of important processing and nutritionalcompounds such as antioxidant flavonoids and carotenoids in fruits andnon-fruit tissues. An example would be potential over-expression inmaize seeds to enhance accumulation of antioxidant compounds fornutritional enhancement of the crop or for extraction.

Finally, expression of NOR gene orthologues (functional equivalents) inother species may regulate maturation of seed pods (which are alsocarpels or “fruits”, for example in soybean, pea, common bean) and/orcereal grains (e.g., rice, maize, wheat, sorghum). Thus over-expressionor repression of the NOR gene may be useful in controlling maturity andmaturation time of various cereals. Protracted or accelerated maturationvia manipulation of the NOR gene may additionally impact qualitycharacteristics such as total protein content, carbohydrate loading,nutritional composition (e.g., via altered levels of carotenoids such asbeta-carotene and lycopene) and total yield.

In support of this example, the inventors have developed T-DNAconstructs (FIG. 1) for altering expression of the NOR gene and havetransformed such constructs into normal and mutant tomato genotypes.FIG. 2 shows that delivery of the normal Nor allele into the genome ofmutant plants results in conversion of fruit from unripe to ripe andresults in a range of degrees of ripening and pigment accumulation.

2. Control of Senescence

The NOR gene controls fruit senescence as demonstrated by the lack ofsenescence in tomato fruits harboring the nor mutation. Senescence ortissue death is thus clearly regulated by NOR in fruit and may bemanipulated in non-fruit tissues via regulated expression of the NORgene. Examples of use may include late fruit-ripening repression inbanana or other tropical or sub-tropical fruits subject to rapid decayto permit desirable ripening but not advanced tissue damage reducingfruit quality and desirability. Over-expression in anthers may result insenescence yielding male-sterility, while if this gene is normallyexpressed in other senescing tissues, gene repression may be useful toinhibit senescence for example in vegetables (spinach, lettuce, cabbage,broccoli).

Studies of the mutant nor phenotype have shown that the nor mutationeffects fruit senescence. The inventor's studies comprising the cloningof the NOR gene and development and observation of transgenic tomatoesconfirm that the NOR gene confers regulation of fruit ripening andsenescence (FIG. 2), and suggest the use of NOR gene nucleic acidcompositions for modification of fruit senescence.

3. Control of Pathogen Infection.

Fruit tissue from nor mutant tomato plants are highly resistant toinfection by opportunistic microbial pathogens (Tigchellar et al.,1978). Post-ripening repression (antisense or co-suppression) of thegene in tomato, or other species (apple, pear, peach, strawberry,citrus. etc), could thus be useful in inhibiting subsequentover-ripening and pathogen susceptibility of fruit. Along these samelines, activity of the NOR gene may participate in non-fruit pathogenresistance for example via repression of low-level of tissue or cellspecific expression in response to pathogen attack. Consequently NORgene repression may thus be used to provide a positive impact onpathogen resistance in fruit and non-fruit tissues.

4. Control of Ethylene Response

Again, phenotypic studies of fruit ripening effects of the nor mutationand the transgenic complementation studies of the inventor's (FIG. 2)demonstrate that the NOR gene influences both ethylene biosynthesis andresponse in fruits. As such, NOR can be utilized to manipulate ripeningand quality as described above. Nevertheless, ethylene impacts numerousaspects of plant growth and development in addition to ripening, asmentioned and referenced herein above. It is important to note here thatinducible over-expression or repression of the NOR gene may be useful incontrolling ethylene responses including abscission, senescence,pathogen resistance, germination, and general stress responses (drought,temperature, water, mechanical damage) leading to increased yield andcrop performance. Specific examples of use might include 1) synchronizedand controlled maturation of cereal grains via high level NOR expressionlate in seed development, 2) high level expression of NOR later in thegrowing season to induce senescence and defoliation of cotton viaover-expression in leaves prior to boll harvest, and 3) synchronized andaccelerated or protracted maturation of seed pods via over-expression orrepression, respectively of NOR in soybean. Finally, as stress responsessuch as responses to pathogen infection, and abiotic stress(temperature, water, mechanical damage) are mediated in part byethylene, over-expression of the NOR gene may positively impact theability of plants to withstand biotic and abiotic insults, thusresulting in enhanced crop performance and yield.

5. DNA Markers for Assisted Breeding

The naturally-occurring nor mutation as stated above is already used inbreeding of fresh market and processing tomatoes. Current phenotypicselection methods require confirmation of genotype at the nor locusthrough analysis of fruit development (i.e., the latest stage of plantdevelopment) with confirmation requiring analysis of subsequent progeny.Such phenotypic screening requires considerable growth space and 2-3months per plant generation cycle.

Isolation of the DNA sequences corresponding to the nor mutation haspermitted development of DNA markers based on sequence variation betweenthe normal versus mutant tomato genotypes. Use of such markers allowsfor definitive genotyping of seedlings in a matter of 1-5 days. Anexample of a DNA marker system based on the nor mutation is shown inFIG. 3. In this example, the 2 bp deletion resulting in the mutation(see below and FIG. 5) is exploited to develop a set of PCR primerswhich distinguishes the normal versus mutant allele. The sequencevariation between the normal versus mutant alleles would be the basisfor development of virtually all types of DNA-based markers fordetermining nor locus genotype through the use of sequences locatedprecisely at (thus 100% accurate) the nor locus.

(iii) Summary

The NOR gene cDNA was identified by the inventors and termed CD-11. Thegene shows similar transcript size in mutant versus normal fruit (FIG.4) though it does show reduced accumulation in the mutant. The inventorshave also shown that the nor mutation results from a 2 bp deletion inthe coding sequence which results in introduction of a premature stopcodon through comparative sequencing of the normal versus mutant allelesof the nor locus (FIG. 5). The NOR gene is related to a family of planttranscription factors associated with multiple aspects of plantdevelopment including meristem and cotyledon development and leafsenescence (Sour et al., 1996; Aida et al., 1997; John et al., 1997).FIG. 6 depicts the DNA sequence NOR cDNA sequence.

The effects of manipulation of the NOR gene in tomato and other plantspecies can be readily anticipated via phenotypic observations of theeffects of the nor mutation on fruit development. In short, it is likelythat most ripening related parameters can be accelerated or inhibited infruit via over-expression or suppression, respectively of NOR. Inaddition it is likely that at least a subset of these effects can alsobe manifested in non-fruit tissues. It would seem particularly likelythat ectopic expression of the NOR gene could bring about effectsassociated with ripening in non-fruit tissues (e.g., senescence,abscission, cell wall alterations and starch conversion, in addition toantioxidant pigment accumulation and associated nutritional enhancement)either directly or in association with other genetic modifications. Ifprocesses such as enhanced disease resistance in non-fruit tissues areinfluenced for example by repression of low level expression of NORgene, then repression of said gene may have a positive impact onenhancing disease resistance as well.

(iv) Conclusion

An important advance of the instant invention is that it provides novelmethods for the modification of plant phenotypes. In particular, byproviding the NOR sequence, the invention allows the creation of plantswith modified phenotypes. The inventors specifically contemplate the useof the NOR sequence, as well as all of the derivatives thereof which areprovided by the invention, to genetically transform plant species forthe purpose of altering plant phenotypes. Exemplary phenotypic effectsare those which are associated with fruit ripening or ethylene response.In particular, the inventors contemplate increasing the expression ofNOR in order to increase fruit ripening and/or ethylene responsiveness,or alternatively, decreasing the effective expression of the NOR gene inorder to delay, protract, and/or inhibited fruit ripening or ethyleneresponses. The expression of NOR sequences in accordance with theinvention may be carried out using the native promoter, oralternatively, promoters that are inducible, viral, synthetic,constitutive as described (Poszkowski et al., 1989; Odell et al., 1985),and temporally regulated, spatially regulated (e.g., tissue-specific),and spatio-temporally regulated (Chau et al., 1989).

Types of effects which could be recognized on fruit ripening include, asdescribed in detail above, processes related to changes in color,texture, flavor, aroma, shelf-life, ethylene responses, nutrientcomposition, cell wall metabolism, and susceptibility to pathogenesisassociated with the ripening process. Similarly, effects on ethyleneresponsiveness which could be effected with the invention include eitherincreased or decreased responsiveness to ethylene. Changes in responseto ethylene may effect fruit ripening, organ abscission, seed or pollendehiscence/shattering, tissue senescence, disease resistance, andresponse to environmental stresses including but not limited to drought,flooding, heat, cold, nutrient deficiency, high or low light intensity,mechanical damage and insect or pathogen infection. Modification of anyof the foregoing effects in a plant, as well as any other effectsassociated with the NOR gene, is specifically contemplated by theinventors and a part of the current invention.

Potentially any method employing the sequences described by theinventors may be used to realize the above-mentioned phenotypic effectsin potentially any plant species, although fruiting effects can beexpected to be realized only in species producing fruit. For example,fruit ripening or ethylene responsiveness could be decreased in a givenplant by transformation of the plant with an expression vectorcomprising an antisense NOR gene. Such a NOR gene could comprise thesequences provided herein, or could represent copies of homologoussequences from other plants isolated using the sequences of theinvention. Decreases in fruit ripening or ethylene responsiveness couldalternatively be realized by use of co-suppression by way ofintroduction of additional NOR sequences into a host genome. In thiscase, for example, by introducing multiple exogenous copies of NORsequences, preferably comprising a functional expression unit,cosuppression of any functional native NOR sequences could be realized,and thereby the phenotype of the plant be modified with respect totraits effected by NOR expression. The effect could be realizedpotentially by use of the NOR promoter, coding sequences or terminators,or using heterologous versions thereof.

In order to realize the phenotypic effects contemplated by theinventors, it is not required that a particular plant be directlytransformed. In particular, once a transgene comprising a sequence ofthe invention has been introduced into a host plant, that transgene maybe passed to any subsequent generation by standard plant breedingprotocols. Such breeding can allow the transgene to be introduced intodifferent lines, preferably of an elite agronomic background, or even todifferent species which can be made sexually compatible with the planthaving the transgene. Breeding protocols may be aided by the use ofgenetic markers which are closely linked to the genes of interest. Assuch, the instant invention extends to any plant which has been directlyintroduced with a transgene prepared in accordance with the invention,or which has received the transgene by way of crossing with a planthaving such a transgene. The invention may additionally be applied toany plant species. Preferably, a plant prepared in accordance with theinvention will be a fruiting plant, for example, tomato, berries such asstrawberries and raspberries, banana, kiwi, avocado, melon, mango,papaya, lychee, pear, stone fruits such as peach, apricot, plum andcherry, in addition to true (anatomical) fruits commonly referred to as“vegetables” including peppers, eggplant, okra, and other non-meloncurcubuts such as cucumber and squash. Specific examples of other plantspecies which could be used in accordance with the invention include,but are not limited to, wheat, maize, rye, rice, turfgrass, oat, barley,sorghum, millet, sugarcane, carrot, tobacco, tomato, potato, soybean,canola, sunflower, alfalfa and cotton.

By way of example, one may utilize an expression vector containing asense or antisense NOR coding region and an appropriate selectablemarker to transform a plant cell of a selected species. Any methodcapable of introducing the expression vector into the cell may be usedin accordance with the invention, for example, use ofAgrobacterium-mediated DNA transfer, microprojectile bombardment, directDNA transfer into pollen, by injection of DNA into reproductive organsof a plant, or by direct injection of DNA into the cells of immatureembryos followed by the rehydration of desiccated embryos, or by directDNA uptake by protoplasted cells. The development or regeneration ofplants containing the foreign, exogenous gene that encodes a polypeptideof interest introduced by Agrobacterium from leaf explants can beachieved by methods well known in the art such as described (Horsch etal., 1985). In this procedure, transformants are cultured in thepresence of a selection agent and in a medium that induces theregeneration of shoots in the plant strain being transformed asdescribed (Fraley et al., 1983). This procedure typically producesshoots within two to four months and those shoots are then transferredto an appropriate root-inducing medium containing the selective agentand an antibiotic to prevent bacterial growth. Shoots that rooted in thepresence of the selective agent to form plantlets are then transplantedto soil or other media to allow the production of roots. Theseprocedures vary depending upon the particular plant strain employed,such variations being well known in the art. By inclusion of aselectable or screenable marker with an expression vector, those cellsreceiving the expression vector may efficiently be isolated from thosethat have not received the vector.

The ultimate goal in the production of transgenic plants having alteredphenotypes is to produce plants which are useful to man. In thisrespect, transgenic plants created in accordance with the currentinvention may be used for virtually any purpose deemed of value to thegrower or to the consumer. For example, the fruit of tomato plants withenhanced fruit ripening characteristics may be harvested and sold toconsumers or used in the production of various food products.Additionally, seed could be harvested from the fruit of a plant preparedin accordance with the instant invention, and the seed may be sold tofarmers for planting in the field or may be directly used as food,either for animals or humans. Alternatively, products may be made fromthe seed itself, for example, oil, starch, pharmaceuticals, and variousindustrial products. Such products may be made from particular plantparts or from the entire plant.

Means for preparing products from plants, such as those that may be madewith the current invention, have been well known since the dawn ofagriculture and will be known to those of skill in the art. Specificmethods for crop utilization may be found in, for example, Sprague andDudley (1988), and Watson and Ramstad (1987).

III. Plant Transformation Constructs

The construction of vectors which may be employed in conjunction withplant transformation techniques according to the invention will be knownto those of skill of the art in light of the present disclosure (see,for example, Sambrook et al., 1989; Gelvin et al., 1990). The techniquesof the current invention are thus not limited to any particular nucleicacid sequences in conjunction with the NOR nucleic acid sequencesprovided herein. Exemplary sequences for use with the invention includethose provided in SEQ ID NO:1, SEQ ID NO:6 and SEQ ID NO:7.

One important use of the sequences of the invention will be in thealteration of plant phenotypes by genetic transformation of plants withsense or antisense NOR genes. The NOR gene may be provided with othersequences. Where an expressible coding region that is not necessarily amarker coding region is employed in combination with a marker codingregion, one may employ the separate coding regions on either the same ordifferent DNA segments for transformation. In the latter case, thedifferent vectors are delivered concurrently to recipient cells tomaximize cotransformation.

The choice of any additional elements used in conjunction with the NORsequences will often depend on the purpose of the transformation. One ofthe major purposes of transformation of crop plants is to addcommercially desirable, agronomically important traits to the plant.Such traits include, but are not limited to, processes related tochanges in fruit color, texture, flavor, aroma, shelf-life, ethyleneresponses, nutrient composition, cell wall metabolism, susceptibility topathogenesis associated with the ripening process, organ abscission,seed or pollen dehiscence/shattering, tissue senescence, diseaseresistance, and response to environmental stresses including but notlimited to drought, flooding, heat, cold, nutrient deficiency, high orlow light intensity, mechanical damage and insect or pathogen infection.In certain embodiments, the present inventors contemplate thetransformation of a recipient cell with more than transformationconstruct. Two or more transgenes can be created in a singletransformation event using either distinct selected-gene encodingvectors, or using a single vector incorporating two or more gene codingsequences.

In other embodiments of the invention, it is contemplated that one maywish to employ replication-competent viral vectors for planttransformation. Such vectors include, for example, wheat dwarf virus(WDV) “shuttle” vectors, such as pW1-11 and PW1-GUS (Ugaki et al.,1991). These vectors are capable of autonomous replication in plantcells as well as E. coli, and as such may provide increased sensitivityfor detecting DNA delivered to transgenic cells. A replicating vectoralso may be useful for delivery of genes flanked by DNA sequences fromtransposable elements such as Ac, Ds, or Mu. It also is contemplatedthat transposable elements would be useful for introducing DNA fragmentslacking elements necessary for selection and maintenance of the plasmidvector in bacteria, e.g., antibiotic resistance genes and origins of DNAreplication. It also is proposed that use of a transposable element suchas Ac, Ds, or Mu would actively promote integration of the desired DNAand hence increase the frequency of stably transformed cells.

It further is contemplated that one may wish to co-transform plants orplant cells with 2 or more vectors. Co-transformation may be achievedusing a vector containing the marker and another gene or genes ofinterest. Alternatively, different vectors, e.g., plasmids, may containthe different genes of interest, and the plasmids may be concurrentlydelivered to the recipient cells. Using this method, the assumption ismade that a certain percentage of cells in which the marker has beenintroduced, also have received the other gene(s) of interest. Thus, notall cells selected by means of the marker, will express the other genesof interest which had been presented to the cells concurrently.

Vectors used for plant transformation may include, for example,plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterialartificial chromosomes) or any other suitable cloning system. Thus whenthe term “vector” or “expression vector” is used, all of the foregoingtypes of vectors, as well as nucleic acid sequences isolated therefrom,are included. It is contemplated that utilization of cloning systemswith large insert capacities will allow introduction of large DNAsequences comprising more than one selected gene. Introduction of suchsequences may be facilitated by use of bacterial or yeast artificialchromosomes (BACs or YACs, respectively), or even plant artificialchromosomes. For example, the use of BACs for Agrobacterium-mediatedtransformation was disclosed by Hamilton et al. (1996).

Particularly useful for transformation are expression cassettes whichhave been isolated from such vectors. DNA segments used for transformingplant cells will, of course, generally comprise the cDNA, gene or geneswhich one desires to introduced into and have expressed in the hostcells. These DNA segments can further include, in addition to a NORcoding sequence, structures such as promoters, enhancers, polylinkers,or even regulatory genes as desired. The DNA segment or gene chosen forcellular introduction will often encode a protein which will beexpressed in the resultant recombinant cells resulting in a screenableor selectable trait and/or which will impart an improved phenotype tothe resulting transgenic plant. However, this may not always be thecase, and the present invention also encompasses transgenic plantsincorporating non-expressed transgenes. Preferred components likely tobe included with vectors used in the current invention are as follows.

(i) Regulatory Elements

The construction of vectors which may be employed in conjunction withthe present invention will be known to those of skill of the art inlight of the present disclosure (see e.g., Sambrook et al., 1989; Gelvinet al., 1990). Preferred constructs will generally include a plantpromoter such as the CaMV 35S promoter (Odell et al., 1985), or otherssuch as CaMV 19S (Lawton et al., 1987), nos (Ebert et al., 1987), Adh(Walker et al., 1987), sucrose synthase (Yang & Russell, 1990),a-tubulin, actin (Wang et al., 1992), cab (Sullivan et al., 1989),PEPCase (Hudspeth & Grula, 1989) or those associated with the R genecomplex (Chandler et al., 1989). Tissue specific promoters such as rootcell promoters (Conkling et al., 1990) and tissue specific enhancers(Fromm et al., 1989) are also contemplated to be particularly useful, asare inducible promoters such as ABA- and turgor-inducible promoters.

As the DNA sequence between the transcription initiation site and thestart of the coding sequence, i.e., the untranslated leader sequence,can influence gene expression, one may also wish to employ a particularleader sequence. Preferred leader sequences are contemplated to includethose which include sequences predicted to direct optimum expression ofthe attached gene, i.e., to include a preferred consensus leadersequence which may increase or maintain mRNA stability and preventinappropriate initiation of translation. The choice of such sequenceswill be known to those of skill in the art in light of the presentdisclosure. Sequences that are derived from genes that are highlyexpressed in plants, and in tomato in particular, will be mostpreferred.

It is contemplated that vectors for use in accordance with the presentinvention may be constructed to include the ocs enhancer element. Thiselement was first identified as a 16 bp palindromic enhancer from theoctopine synthase (ocs) gene of Agrobacterium (Ellis et al., 1987), andis present in at least 10 other promoters (Bouchez et al., 1989). It isproposed that the use of an enhancer element, such as the ocs elementand particularly multiple copies of the element, will act to increasethe level of transcription from adjacent promoters when applied in thecontext of plant transformation.

It is specifically envisioned that NOR coding sequences may beintroduced under the control of novel promoters or enhancers, etc., orperhaps even homologous or tissue specific (e.g., root-, collar/sheath-,whorl-, stalk-, earshank-, kernel- or leaf-specific) promoters orcontrol elements. Indeed, it is envisioned that a particular use of thepresent invention will be the targeting sense or antisense NORexpression in a tissue-specific manner. For example, these sequencescould be targeted to the fruit.

Vectors for use in tissue-specific targeting of genes in transgenicplants will typically include tissue-specific promoters and may alsoinclude other tissue-specific control elements such as enhancersequences. Promoters which direct specific or enhanced expression incertain plant tissues will be known to those of skill in the art inlight of the present disclosure. These include, for example, the rbcSpromoter, specific for green tissue; the ocs, nos and mas promoterswhich have higher activity in roots or wounded leaf tissue; a truncated(−90 to +8) 35S promoter which directs enhanced expression in roots, andan a-tubulin gene that directs expression in roots.

It also is contemplated that tissue specific expression may befunctionally accomplished by introducing a constitutively expressed gene(all tissues) in combination with an antisense gene that is expressedonly in those tissues where the gene product is not desired. Forexample, a gene coding for a NOR sequence may be introduced such that itis expressed in all tissues using the 35S promoter from CauliflowerMosaic Virus. Expression of an antisense transcript of the same NOR genein the fruit of a plant would prevent expression of the NOR gene only inthe fruit.

Alternatively, one may wish to obtain novel tissue-specific promotersequences for use in accordance with the present invention. To achievethis, one may first isolate cDNA clones from the tissue concerned andidentify those clones which are expressed specifically in that tissue,for example, using Northern blotting. Ideally, one would like toidentify a gene that is not present in a high copy number, but whichgene product is relatively abundant in specific tissues. The promoterand control elements of corresponding genomic clones may then belocalized using the techniques of molecular biology known to those ofskill in the art.

It is contemplated that expression of sense or antisense NOR genes intransgenic plants may in some cases be desired only under specifiedconditions. It is contemplated that expression of such sequences at highlevels may have detrimental effects. It is known that a large number ofgenes exist that respond to the environment. For example, expression ofsome genes such as rbcS, encoding the small subunit of ribulosebisphosphate carboxylase, is regulated by light as mediated throughphytochrome. Other genes are induced by secondary stimuli. A number ofgenes have been shown to be induced by ABA (Skriver and Mundy, 1990).Therefore, in particular embodiments, inducible expression of thenucleic acid sequences of the invention may be desired.

It also is contemplated by the inventors that in some embodiments of thepresent invention expression of a NOR gene will be desired only in acertain time period during the development of the plant. Developmentaltiming is frequently correlated with tissue specific gene expression.For example, expression of certain genes associated with fruit ripeningwill only be expressed at certain stages of fruit development.

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This will generally be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit, or signal, peptide willtransport the protein to a particular intracellular, or extracellulardestination, respectively, and will then be post-translationallyremoved. Transit or signal peptides act by facilitating the transport ofproteins through intracellular membranes, e.g., vacuole, vesicle,plastid and mitochondrial membranes, whereas signal peptides directproteins through the extracellular membrane.

A particular example of such a use concerns the direction of a herbicideresistance selectable marker gene, such as the EPSPS gene, to aparticular organelle such as the chloroplast rather than to thecytoplasm. This is exemplified by the use of the rbcS transit peptidewhich confers plastid-specific targeting of proteins. In addition, it isproposed that it may be desirable to target NOR genes to theextracellular spaces or to the vacuole.

It also is contemplated that it may be useful to target DNA itselfwithin a cell. For example, it may be useful to target introduced DNA tothe nucleus as this may increase the frequency of transformation. Withinthe nucleus itself it would be useful to target a gene in order toachieve site specific integration. For example, it would be useful tohave an gene introduced through transformation replace an existing genein the cell.

(ii) Terminators

Transformation constructs prepared in accordance with the invention willtypically include a 3′ end DNA sequence that acts as a signal toterminate transcription and allow for the poly-adenylation of the mRNAproduced by coding sequences operably linked to a NOR gene. In oneembodiment of the invention, the native NOR gene is used. Alternatively,a heterologous 3′ end may enhance the expression of sense or antisenseNOR sequences. Terminators which are deemed to be particularly useful inthis context include those from the nopaline synthase gene ofAgrobacterium tumefaciens (nos 3′ end) (Bevan et al., 1983), theterminator for the T7 transcript from the octopine synthase gene ofAgrobacterium tumefaciens, and the 3′ end of the protease inhibitor I orII genes from potato or tomato. Regulatory elements such as Adh intron(Callis et al., 1987), sucrose synthase intron (Vasil et al., 1989) orTMV omega element (Gallie et at, 1989), may further be included wheredesired.

(iii) Transit or Signal Peptides

Sequences that are joined to the coding sequence of an expressed gene,which are removed post-translationally from the initial translationproduct and which facilitate the transport of the protein into orthrough intracellular or extracellular membranes, are termed transit(usually into vacuoles, vesicles, plastids and other intracellularorganelles) and signal sequences (usually to the endoplasmic reticulum,golgi apparatus and outside of the cellular membrane). By facilitatingthe transport of the protein into compartments inside and outside thecell, these sequences may increase the accumulation of gene productprotecting them from proteolytic degradation. These sequences also allowfor additional mRNA sequences from highly expressed genes to be attachedto the coding sequence of the genes. Since mRNA being translated byribosomes is more stable than naked mRNA, the presence of translatablemRNA in front of the gene may increase the overall stability of the mRNAtranscript from the gene and thereby increase synthesis of the geneproduct. Since transit and signal sequences are usuallypost-translationally removed from the initial translation product, theuse of these sequences allows for the addition of extra translatedsequences that may not appear on the final polypeptide. It further iscontemplated that targeting of certain proteins may be desirable inorder to enhance the stability of the protein (U.S. Pat. No. 5,545,818,incorporated herein by reference in its entirety).

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This generally will be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit, or signal, peptide willtransport the protein to a particular intracellular, or extracellulardestination, respectively, and will then be post-translationallyremoved.

(iv) Marker Genes

By employing a selectable or screenable marker protein, one can provideor enhance the ability to identify transformants. “Marker genes” aregenes that impart a distinct phenotype to cells expressing the markerprotein and thus allow such transformed cells to be distinguished fromcells that do not have the marker. Such genes may encode either aselectable or screenable marker, depending on whether the marker confersa trait which one can “select” for by chemical means, i.e., through theuse of a selective agent (e.g., a herbicide, antibiotic, or the like),or whether it is simply a trait that one can identify throughobservation or testing, i.e., by “screening”′ (e.g., the greenfluorescent protein). Of course, many examples of suitable markerproteins are known to the art and can be employed in the practice of theinvention.

Included within the terms selectable or screenable markers also aregenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which are secretable antigens that can be identified byantibody interaction, or even secretable enzymes which can be detectedby their catalytic activity. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA; small active enzymes detectable in extracellular solution (e.g.,α-amylase, β-lactamase, phosphinothricin acetyltransferase); andproteins that are inserted or trapped in the cell wall (e.g., proteinsthat include a leader sequence such as that found in the expression unitof extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a protein that becomes sequestered in the cell wall, and whichprotein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

1. Selectable Markers

Many selectable marker coding regions may be used in connection with theNOR sequences of the present invention including, but not limited to,neo (Potrykus et al., 1985), which provides kanamycin resistance and canbe selected for using kanamycin, G418, paromomycin, etc.; bar, whichconfers bialaphos or phosphinothricin resistance; a mutant EPSP synthaseprotein (Hinchee et al., 1988) conferring glyphosate resistance; anitrilase such as bxn from Klebsiella ozaenae which confers resistanceto bromoxynil (Stalker et al., 1988); a mutant acetolactate synthase(ALS) which confers resistance to imidazolinone, sulfonylurea or otherALS inhibiting chemicals (European Patent Application 154,204, 1985); amethotrexate resistant DHFR (Thillet et al., 1988), a dalapondehalogenase that confers resistance to the herbicide dalapon; or amutated anthranilate synthase that confers resistance to 5-methyltryptophan. Where a mutant EPSP synthase is employed, additional benefitmay be realized through the incorporation of a suitable chloroplasttransit peptide, CTP (U.S. Pat. No. 5,188,642) or OTP (U.S. Pat. No.5,633,448) and use of a modified maize EPSPS (PCT Application WO97/04103).

An illustrative embodiment of selectable marker capable of being used insystems to select transformants are those that encode the enzymephosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes. The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami etal., 1986; Twell et al., 1989) causing rapid accumulation of ammonia andcell death.

Where one desires to employ a bialaphos resistance gene in the practiceof the invention, the inventor has discovered that particularly usefulgenes for this purpose are the bar or pat genes obtainable from speciesof Streptomyces (e.g., ATCC No. 21,705). The cloning of the bar gene hasbeen described (Murakami et al., 1986; Thompson et al., 1987) as has theuse of the bar gene in the context of plants (De Block et al., 1987; DeBlock et al., 1989; U.S. Pat. No. 5,550,318).

2. Screenable Markers

Screenable markers that may be employed include a β-glucuronidase (GUS)or uidA gene which encodes an enzyme for which various chromogenicsubstrates are known; an R-locus gene, which encodes a product thatregulates the production of anthocyanin pigments (red color) in planttissues (Dellaporta et al., 1988); a β-lactamase gene (Sutcliffe, 1978),which encodes an enzyme for which various chromogenic substrates areknown (e.g., PADAC, a chromogenic cephalosporin); a xyle gene (Zukowskyet al., 1983) which encodes a catechol dioxygenase that can convertchromogenic catechols; an α-amylase gene (Ikuta et al., 1990); atyrosinase gene (Katz et al., 1983) which encodes an enzyme capable ofoxidizing tyrosine to DOPA and dopaquinone which in turn condenses toform the easily-detectable compound melanin; a β-galactosidase gene,which encodes an enzyme for which there are chromogenic substrates; aluciferase (lux) gene (Ow et al., 1986), which allows forbioluminescence detection; an aequorin gene (Prasher et al., 1985) whichmay be employed in calcium-sensitive bioluminescence detection; or agene encoding for green fluorescent protein (Sheen et al., 1995;Haseloff et al., 1997; Reichel et al., 1996; Tian et al., 1997; WO97/41228).

Genes from the maize R gene complex can also be used as screenablemarkers. The R gene complex in maize encodes a protein that acts toregulate the production of anthocyanin pigments in most seed and planttissue. Maize strains can have one, or as many as four, R alleles whichcombine to regulate pigmentation in a developmental and tissue specificmanner. Thus, an R gene introduced into such cells will cause theexpression of a red pigment and, if stably incorporated, can be visuallyscored as a red sector. If a maize line carries dominant alleles forgenes encoding for the enzymatic intermediates in the anthocyaninbiosynthetic pathway (C2, A1, A2, Bz1 and Bz2), but carries a recessiveallele at the R locus, transformation of any cell from that line with Rwill result in red pigment formation. Exemplary lines include Wisconsin22 which contains the rg-Stadler allele and TR112, a K55 derivativewhich is r-g, b, Pl. Alternatively, any genotype of maize can beutilized if the C1 and R alleles are introduced together.

Another screenable marker contemplated for use in the present inventionis firefly luciferase, encoded by the lux gene. The presence of the luxgene in transformed cells may be detected using, for example, X-rayfilm, scintillation counting, fluorescent spectrophotometry, low-lightvideo cameras, photon counting cameras or multiwell luminometry. It alsois envisioned that this system may be developed for populationalscreening for bioluminescence, such as on tissue culture plates, or evenfor whole plant screening. The gene which encodes green fluorescentprotein (GFP) is contemplated as a particularly useful reporter gene(Sheen et al., 1995; Haseloff et al., 1997; Reichel et al., 1996; Tianet al., 1997; WO 97/41228). Expression of green fluorescent protein maybe visualized in a cell or plant as fluorescence following illuminationby particular wavelengths of light. Where use of a screenable markergene such as lux or GFP is desired, the inventors contemplated thatbenefit may be realized by creating a gene fusion between the screenablemarker gene and a selectable marker gene, for example, a GFP-NPTII genefusion. This could allow, for example, selection of transformed cellsfollowed by screening of transgenic plants or seeds.

3. Negative Selectable Markers

Introduction of genes encoding traits that can be selected against maybe useful for eliminating undesirable linked genes. It is contemplatedthat when two or more genes are introduced together by cotransformationthat the genes will be linked together on the host chromosome. Forexample, a gene encoding Bt that confers insect resistance on the plantmay be introduced into a plant together with a bar gene that is usefulas a selectable marker and confers resistance to the herbicide Liberty®on the plant. However, it may not be desirable to have an insectresistant plant that also is resistant to the herbicide Liberty®. It isproposed that one also could introduce an antisense bar gene that isexpressed in those tissues where one does not want expression of the bargene, e.g., in whole plant parts. Hence, although the bar gene isexpressed and is useful as a selectable marker, it is not useful toconfer herbicide resistance on the whole plant. The bar antisense geneis a negative selectable marker.

It also is contemplated that negative selection is necessary in order toscreen a population of transformants for rare homologous recombinantsgenerated through gene targeting. For example, a homologous recombinantmay be identified through the inactivation of a gene that was previouslyexpressed in that cell. The antisense gene to neomycinphosphotransferase II (NPT II) has been investigated as a negativeselectable marker in tobacco (Nicotiana tabacum) and Arabidopsisthaliana (Xiang. and Guerra, 1993). In this example, both sense andantisense NPT II genes are introduced into a plant throughtransformation and the resultant plants are sensitive to the antibiotickanamycin. An introduced gene that integrates into the host cellchromosome at the site of the antisense NPT II gene, and inactivates theantisense gene, will make the plant resistant to kanamycin and otheraminoglycoside antibiotics. Therefore, rare, site-specific recombinantsmay be identified by screening for antibiotic resistance. Similarly, anygene, native to the plant or introduced through transformation, thatwhen inactivated confers resistance to a compound, may be useful as anegative selectable marker.

It is contemplated that negative selectable markers also may be usefulin other ways. One application is to construct transgenic lines in whichone could select for transposition to unlinked sites. In the process oftagging it is most common for the transposable element to move to agenetically linked site on the same chromosome. A selectable marker forrecovery of rare plants in which transposition has occurred to anunlinked locus would be useful. For example, the enzyme cytosinedeaminase may be useful for this purpose (Stouggard, 1993). In thepresence of this enzyme the compound 5-fluorocytosine is converted to5-fluorouracil which is toxic to plant and animal cells. If atransposable element is linked to the gene for the enzyme cytosinedeaminase, one may select for transposition to unlinked sites byselecting for transposition events in which the resultant plant is nowresistant to 5-fluorocytosine. The parental plants and plants containingtranspositions to linked sites will remain sensitive to5-fluorocytosine. Resistance to 5-fluorocytosine is due to loss of thecytosine deaminase gene through genetic segregation of the transposableelement and the cytosine deaminase gene. Other genes that encodeproteins that render the plant sensitive to a certain compound will alsobe useful in this context. For example, T-DNA gene 2 from Agrobacteriumtumefaciens encodes a protein that catalyzes the conversion ofα-naphthalene acetamide (NAM) to α-naphthalene acetic acid (NAA) rendersplant cells sensitive to high concentrations of NAM (Depicker et al.,1988).

It also is contemplated that negative selectable markers may be usefulin the construction of transposon tagging lines. For example, by markingan autonomous transposable element such as Ac, Master Mu, or En/Spn witha negative selectable marker, one could select for transformants inwhich the autonomous element is not stably integrated into the genome.It is proposed that this would be desirable, for example, when transientexpression of the autonomous element is desired to activate in trans thetransposition of a defective transposable element, such as Ds, butstable integration of the autonomous element is not desired. Thepresence of the autonomous element may not be desired in order tostabilize the defective element, i.e., prevent it from furthertransposing. However, it is proposed that if stable integration of anautonomous transposable element is desired in a plant the presence of anegative selectable marker may make it possible to eliminate theautonomous element during the breeding process.

(iv) Ribozymes

DNA may be introduced into plants for the purpose of expressing RNAtranscripts that function to affect plant phenotype yet are nottranslated into protein. Two examples are antisense RNA, which isdiscussed in detail below, and RNA with ribozyme activity. Both mayserve possible functions in reducing or eliminating expression of nativeor introduced plant genes, for example, a NOR gene. However, as detailedbelow, DNA need not be expressed to effect the phenotype of a plant.Genes also may be constructed or isolated, which when transcribed,produce RNA enzymes (ribozymes) which can act as endoribonucleases andcatalyze the cleavage of RNA molecules with selected sequences. Thecleavage of selected messenger RNAs can result in the reduced productionof their encoded polypeptide products. These genes may be used toprepare novel transgenic plants which possess them. The transgenicplants may possess reduced levels of polypeptides including, but notlimited to, the polypeptides cited above.

Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987;Forster and Symons, 1987). For example, a large number of ribozymesaccelerate phosphoester transfer reactions with a high degree ofspecificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes.

Several different ribozyme motifs have been described with RNA cleavageactivity (Symons, 1992). Examples include sequences from the Group Iself splicing introns including Tobacco Ringspot Virus (Prody et al.,1986), Avocado Sunblotch Viroid (Palukaitis et al., 1979), and LucerneTransient Streak Virus (Forster and Symons, 1987). Sequences from theseand related viruses are referred to as hammerhead ribozyme based on apredicted folded secondary structure.

Other suitable ribozymes include sequences from RNase P with RNAcleavage activity (Yuan et al., 1992, Yuan and Altman, 1994, U.S. Pat.Nos. 5,168,053 and 5,624,824), hairpin ribozyme structures(Berzal-Herranz et al., 1992; Chowrira et al., 1994) and Hepatitis Deltavirus based ribozymes (U.S. Pat. No. 5,625,047). The general design andoptimization of ribozyme directed RNA cleavage activity has beendiscussed in detail (Haseloff and Gerlach, 1988, Symons, 1992, Chowriraet al., 1994; Thompson et al., 1995).

The other variable on ribozyme design is the selection of a cleavagesite on a given target RNA. Ribozymes are targeted to a given sequenceby virtue of annealing to a site by complimentary base pairinteractions. Two stretches of homology are required for this targeting.These stretches of homologous sequences flank the catalytic ribozymestructure defined above. Each stretch of homologous sequence can vary inlength from 7 to 15 nucleotides. The only requirement for defining thehomologous sequences is that, on the target RNA, they are separated by aspecific sequence which is the cleavage site. For hammerhead ribozyme,the cleavage site is a dinucleotide sequence on the target RNA is auracil (U) followed by either an adenine, cytosine or uracil (A, C or U)(Perriman et al., 1992; Thompson et al., 1995). The frequency of thisdinucleotide occurring in any given RNA is statistically 3 out of 16.Therefore, for a given target messenger RNA of 1000 bases, 187dinucleotide cleavage sites are statistically possible.

Designing and testing ribozymes for efficient cleavage of a target RNAis a process well known to those skilled in the art. Examples ofscientific methods for designing and testing ribozymes are described byChowrira et al., (1994) and Lieber and Strauss (1995), each incorporatedby reference. The identification of operative and preferred sequencesfor use in down regulating a given gene is simply a matter of preparingand testing a given sequence, and is a routinely practiced “screening”method known to those of skill in the art.

(v) Induction of Gene Silencing

It also is possible that genes may be introduced to produce noveltransgenic plants which have reduced expression of a native gene productby the mechanism of co-suppression, thus this technique could be used inaccordance with the invention. It has been demonstrated in tobacco,tomato, and petunia (Goring et al., 1991; Smith et al., 1990; Napoli etal., 1990; van der Krol et al., 1990) that expression of the sensetranscript of a native gene will reduce or eliminate expression of thenative gene in a manner similar to that observed for antisense genes.The introduced gene may encode all or part of the targeted nativeprotein but its translation may not be required for reduction of levelsof that native protein.

IV. Antisense Constructs

Antisense treatments are one way of altering fruit quality and/orethylene response and the characteristics associated therewith inaccordance with the invention. In particular, constructs comprising theNOR gene in antisense orientation may be used to decrease or effectivelyeliminate the expression of the gene in a plant. As such, antisensetechnology may be used to “knock-out” the function of a NOR gene orhomologous sequences thereof, thereby causing the delay, protraction orinhibition of fruit ripening and/or decreased ethylene responsiveness,as well as the effects associated therewith.

Antisense methodology takes advantage of the fact that nucleic acidstend to pair with “complementary” sequences. By complementary, it ismeant that polynucleotides are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,the larger purines will base pair with the smaller pyrimidines to formcombinations of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. Inclusion of less common bases such asinosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNA's, may be employed to inhibit gene transcription or translation orboth within a host cell, either in vitro or in vivo, such as within ahost animal, including a human subject.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructswill include regions complementary to intron/exon splice junctions.Thus, it is proposed that a preferred embodiment includes an antisenseconstruct with complementarity to regions within 50-200 bases of anintron-exon splice junction. It has been observed that some exonsequences can be included in the construct without seriously affectingthe target selectivity thereof. The amount of exonic material includedwill vary depending on the particular exon and intron sequences used.One can readily test whether too much exon DNA is included simply bytesting the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

As stated above, “complementary” or “antisense” means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme; see above) could be designed. These molecules, thoughhaving less than 50% homology, would bind to target sequences underappropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

V. Methods for Plant Transformation

Suitable methods for plant transformation for use with the currentinvention are believed to include virtually any method by which DNA canbe introduced into a cell, such as by direct delivery of DNA such as byPEG-mediated transformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake (Potrykus et al, 1985), byelectroporation (U.S. Pat. No. 5,384,253, specifically incorporatedherein by reference in its entirety), by agitation with silicon carbidefibers (Kaeppler et al., 1990; U.S. Pat. No. 5,302,523, specificallyincorporated herein by reference in its entirety; and U.S. Pat. No.5,464,765, specifically incorporated herein by reference in itsentirety), by Agrobacterium-mediated transformation (U.S. Pat. No.5,591,616 and U.S. Pat. No. 5,563,055; both specifically incorporatedherein by reference) and by acceleration of DNA coated particles (U.S.Pat. No. 5,550,318; U.S. Pat. No. 5,538,877; and U.S. Pat. No.5,538,880; each specifically incorporated herein by reference in itsentirety), etc. Through the application of techniques such as these, thecells of virtually any plant species may be stably transformed, andthese cells developed into transgenic plants.

(i) Agrobacterium-Mediated Transformation

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described by Fraley etal., (1985), Rogers et al., (1987) and U.S. Pat. No. 5,563,055,specifically incorporated herein by reference in its entirety.

Agrobacterium-mediated transformation is most efficient indicotyledonous plants and is the preferable method for transformation ofdicots, including Arabidopsis, tobacco, tomato, and potato. Indeed,while Agrobacterium-mediated transformation has been routinely used withdicotyledonous plants for a number of years, it has only recently becomeapplicable to monocotyledonous plants. Advances inAgrobacterium-mediated transformation techniques have now made thetechnique applicable to nearly all monocotyledonous plants. For example,Agrobacterium-mediated transformation techniques have now been appliedto rice (Hiei et al., 1997; U.S. Pat. No. 5,591,616, specificallyincorporated herein by reference in its entirety), wheat (McCormac etal., 1998), barley (Tingay et al., 1997; McCormac et al., 1998), andmaize (Ishidia et al., 1996).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described(Rogers et al., 1987) have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Inaddition, Agrobacterium containing both armed and disarmed Ti genes canbe used for the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

(ii) Electroporation

Where one wishes to introduce DNA by means of electroporation, themethod of Krzyzek et al. (U.S. Pat. No. 5,384,253, incorporated hereinby reference in its entirety) may be particularly advantageous. In thismethod, certain cell wall-degrading enzymes, such as pectin-degradingenzymes, are employed to render the target recipient cells moresusceptible to transformation by electroporation than untreated cells.Alternatively, recipient cells are made more susceptible totransformation by mechanical wounding.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wounding in acontrolled manner. Examples of some species which have been transformedby electroporation of intact cells include maize (U.S. Pat. No.5,384,253; Rhodes et al., 1995; D'Halluin et al., 1992), wheat (Zhou etal., 1993), tomato (Hou and Lin, 1996), soybean (Christou et al., 1987)and tobacco (Lee et al., 1989).

One also may employ protoplasts for electroporation transformation ofplants (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon-derivedprotoplasts is described by Dhir and Widholm in Intl. Patent Appl. Publ.No. WO 9217598 (specifically incorporated herein by reference). Otherexamples of species for which protoplast transformation has beendescribed include barley (Lazerri, 1995), sorghum (Battraw et al.,1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) andtomato (Tsukada, 1989).

(iii) Microprojectile Bombardment

Another method for delivering transforming DNA segments to plant cellsin accordance with the invention is microprojectile bombardment (U.S.Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No. 5,610,042;and PCT Application WO 94/09699; each of which is specificallyincorporated herein by reference in its entirety). In this method,particles may be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, platinum, and preferably, gold. It is contemplated that insome instances DNA precipitation onto metal particles would not benecessary for DNA delivery to a recipient cell using microprojectilebombardment. However, it is contemplated that particles may contain DNArather than be coated with DNA. Hence, it is proposed that DNA-coatedparticles may increase the level of DNA delivery via particlebombardment but are not, in and of themselves, necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a filter surfacecovered with monocot plant cells cultured in suspension. The screendisperses the particles so that they are not delivered to the recipientcells in large aggregates. It is believed that a screen interveningbetween the projectile apparatus and the cells to be bombarded reducesthe size of projectiles aggregate and may contribute to a higherfrequency of transformation by reducing the damage inflicted on therecipient cells by projectiles that are too large.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. Examples of species forwhich have been transformed by microprojectile bombardment includemonocot species such as maize (PCT Application WO 95/06128), barley(Ritala et al., 1994; Hensgens et al., 1993), wheat (U.S. Pat. No.5,563,055, specifically incorporated herein by reference in itsentirety), rice (Hensgens et al., 1993), oat (Torbet et al., 1995;Torbet et al., 1998), rye (Hensgens et al., 1993), sugarcane (Bower etal., 1992), and sorghum (Casa et al., 1993; Hagio et al., 1991); as wellas a number of dicots including tobacco (Tomes et al., 1990; Buising andBenbow, 1994), soybean (U.S. Pat. No. 5,322,783, specificallyincorporated herein by reference in its entirety), sunflower (Knittel etal. 1994), peanut (Singsit et al., 1997), cotton (McCabe and Martinell,1993), tomato (VanEck et al. 1995), and legumes in general (U.S. Pat.No. 5,563,055, specifically incorporated herein by reference in itsentirety).

(iv) Other Transformation Methods

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Omirulleh et al., 1993; Frommet al., 1986; Uchimiya et al., 1986; Callis et al., 1987; Marcotte etal., 1988).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastshave been described (Fujimara et al., 1985; Toriyama et al., 1986;Yamada et al., 1986; Abdullah et al., 1986; Omirulleh et al, 1993 andU.S. Pat. No. 5,508,184; each specifically incorporated herein byreference in its entirety). Examples of the use of direct uptaketransformation of cereal protoplasts include transformation of rice(Ghosh-Biswas et al., 1994), sorghum (Battraw and Hall, 1991), barley(Lazerri, 1995), oat (Zheng and Edwards, 1990) and maize (Omirulleh etal., 1993).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, 1989). Also,silicon carbide fiber-mediated transformation may be used with orwithout protoplasting (Kaeppler, 1990; Kaeppler et al., 1992; U.S. Pat.No. 5,563,055, specifically incorporated herein by reference in itsentirety). Transformation with this technique is accomplished byagitating silicon carbide fibers together with cells in a DNA solution.DNA passively enters as the cell are punctured. This technique has beenused successfully with, for example, the monocot cereals maize (PCTApplication WO 95/06128, specifically incorporated herein by referencein its entirety; Thompson, 1995) and rice (Nagatani, 1997).

VI. Site Specific Integration or Excision of Transgenes

It is specifically contemplated by the inventors that one could employtechniques for the site-specific integration or excision oftransformation constructs prepared in accordance with the instantinvention. Alternatively, site-specific integration techniques could beused to insertionally mutagenize or replace a native NOR gene sequence.An advantage of site-specific integration or excision is that it can beused to overcome problems associated with conventional transformationtechniques, in which transformation constructs typically randomlyintegrate into a host genome in multiple copies. This random insertionof introduced DNA into the genome of host cells can be lethal if theforeign DNA inserts into an essential gene. In addition, the expressionof a transgene may be influenced by “position effects” caused by thesurrounding genomic DNA. Further, because of difficulties associatedwith plants possessing multiple transgene copies, including genesilencing, recombination and unpredictable inheritance, it is typicallydesirable to control the copy number of the inserted DNA, often onlydesiring the insertion of a single copy of the DNA sequence.

Site-specific integration or excision of transgenes or parts oftransgenes can be achieved in plants by means of homologousrecombination (see, for example, U.S. Pat. No. 5,527,695, specificallyincorporated herein by reference in its entirety). Homologousrecombination is a reaction between any pair of DNA sequences having asimilar sequence of nucleotides, where the two sequences interact(recombine) to form a new recombinant DNA species. The frequency ofhomologous recombination increases as the length of the sharednucleotide DNA sequences increases, and is higher with linearizedplasmid molecules than with circularized plasmid molecules. Homologousrecombination can occur between two DNA sequences that are less thanidentical, but the recombination frequency declines as the divergencebetween the two sequences increases.

Introduced DNA sequences can be targeted via homologous recombination bylinking a DNA molecule of interest to sequences sharing homology withendogenous sequences of the host cell. For example, conserved NORsequences could be used to replace a native NOR sequence with one of theNOR sequences provided herein. Once the DNA enters the cell, the twohomologous sequences can interact to insert the introduced DNA at thesite where the homologous genomic DNA sequences were located. Therefore,the choice of homologous sequences contained on the introduced DNA willdetermine the site where the introduced DNA is integrated via homologousrecombination. For example, if the DNA sequence of interest is linked toDNA sequences sharing homology to a single copy gene of a host plantcell, the DNA sequence of interest will be inserted via homologousrecombination at only that single specific site. However, if the DNAsequence of interest is linked to DNA sequences sharing homology to amulticopy gene of the host eukaryotic cell, then the DNA sequence ofinterest can be inserted via homologous recombination at each of thespecific sites where a copy of the gene is located.

DNA can be inserted into the host genome by a homologous recombinationreaction involving either a single reciprocal recombination (resultingin the insertion of the entire length of the introduced DNA) or througha double reciprocal recombination (resulting in the insertion of onlythe DNA located between the two recombination events). For example, ifone wishes to insert a foreign gene into the genomic site where aselected gene is located, the introduced DNA should contain sequenceshomologous to the selected gene. A single homologous recombination eventwould then result in the entire introduced DNA sequence being insertedinto the selected gene. Alternatively, a double recombination event canbe achieved by flanking each end of the DNA sequence of interest (thesequence intended to be inserted into the genome) with DNA sequenceshomologous to the selected gene. A homologous recombination eventinvolving each of the homologous flanking regions will result in theinsertion of the foreign DNA. Thus only those DNA sequences locatedbetween the two regions sharing genomic homology become integrated intothe genome.

Although introduced sequences can be targeted for insertion into aspecific genomic site via homologous recombination, in higher eukaryoteshomologous recombination is a relatively rare event compared to randominsertion events. In plant cells, foreign DNA molecules find homologoussequences in the cell's genome and recombine at a frequency ofapproximately 0.5-4.2×10⁻⁴. Thus any transformed cell that contains anintroduced DNA sequence integrated via homologous recombination willalso likely contain numerous copies of randomly integrated introducedDNA sequences. Therefore, to maintain control over the copy number andthe location of the inserted DNA, these randomly inserted DNA sequencescan be removed. One manner of removing these random insertions is toutilize a site-specific recombinase system. In general, a site specificrecombinase system consists of three elements: two pairs of DNA sequence(the site-specific recombination sequences) and a specific enzyme (thesite-specific recombinase). The site-specific recombinase will catalyzea recombination reaction only between two site-specific recombinationsequences.

A number of different site specific recombinase systems could beemployed in accordance with the instant invention, including, but notlimited to, the Cre/lox system of bacteriophage P1 (U.S. Pat. No.5,658,772, specifically incorporated herein by reference in itsentirety), the FLP/FRT system of yeast (Golic and Lindquist, 1989), theGin recombinase of phage Mu (Maeser et al., 1991), the Pin recombinaseof E. coli (Enomoto et al., 1983), and the R/RS system of the pSR1plasmid (Araki et al., 1992). The bacteriophage P1 Cre/lox and the yeastFLP/FRT systems constitute two particularly useful systems for sitespecific integration or excision of transgenes. In these systems arecombinase (Cre or FLP) will interact specifically with its respectivesite-specific recombination sequence (lox or FRT, respectively) toinvert or excise the intervening sequences. The sequence for each ofthese two systems is relatively short (34 bp for lox and 47 bp for FRT)and therefore, convenient for use with transformation vectors.

Experiments on the performance of the FLP/FRT system in both maize andrice protoplasts indicate that FRT site structure, and amount of the FLPprotein present, affects excision activity. In general, short incompleteFRT sites leads to higher accumulation of excision products than thecomplete full-length FRT sites. The systems can catalyze both intra- andintermolecular reactions in maize protoplasts, indicating its utilityfor DNA excision as well as integration reactions. The recombinationreaction is reversible and this reversibility can compromise theefficiency of the reaction in each direction. Altering the structure ofthe site-specific recombination sequences is one approach to remedyingthis situation. The site-specific recombination sequence can be mutatedin a manner that the product of the recombination reaction is no longerrecognized as a substrate for the reverse reaction, thereby stabilizingthe integration or excision event.

In the Cre-lox system, discovered in bacteriophage P1, recombinationbetween loxP sites occurs in the presence of the Cre recombinase (see,e.g., U.S. Pat. No. 5,658,772, specifically incorporated herein byreference in its entirety). This system has been utilized to excise agene located between two lox sites which had been introduced into ayeast genome (Sauer, 1987). Cre was expressed from an inducible yeastGAL1 promoter and this Cre gene was located on an autonomouslyreplicating yeast vector.

Since the lox site is an asymmetrical nucleotide sequence, lox sites onthe same DNA molecule can have the same or opposite orientation withrespect to each other. Recombination between lox sites in the sameorientation results in a deletion of the DNA segment located between thetwo lox sites and a connection between the resulting ends of theoriginal DNA molecule. The deleted DNA segment forms a circular moleculeof DNA. The original DNA molecule and the resulting circular moleculeeach contain a single lox site. Recombination between lox sites inopposite orientations on the same DNA molecule results in an inversionof the nucleotide sequence of the DNA segment located between the twolox sites. In addition, reciprocal exchange of DNA segments proximate tolox sites located on two different DNA molecules can occur. All of theserecombination events are catalyzed by the product of the Cre codingregion.

VI. Biological Functional Equivalents

Modification and changes may be made in the nucleic acids provided bythe present invention and accordingly the structure of the polypeptidesencoded thereby, and still obtain functional molecules that encode a NORpolypeptide. The following is a discussion based upon alerting nucleicacids in the NOR sequences to result in a changing of the amino acids ofa NOR polypeptide to create an equivalent, or even an improved,second-generation molecule. In particular embodiments of the invention,mutated NOR proteins are contemplated to be useful for increasing theactivity of the protein. The amino acid changes may be achieved bychanging the codons of the DNA sequence, according to the codons givenin Table 1. TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte et al., 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte et al., 1982),these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Another embodiment for the preparation of polypeptides according to theinvention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure. See, for example, Johnson et al., “Peptide Turn Mimetics” inBIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, NewYork (1993). The underlying rationale behind the use of peptide mimeticsis that the peptide backbone of proteins exists chiefly to orient aminoacid side chains in such a way as to facilitate molecular interactions,such as those of antibody and antigen. A peptide mimetic is expected topermit molecular interactions similar to the natural molecule. Theseprinciples may be used, in conjunction with the principles outlineabove, to engineer second generation molecules having many of thenatural properties of the starting gene product, but with altered andeven improved characteristics.

VIII. Production and Characterization of Stably Transformed Plants

After effecting delivery of exogenous DNA to recipient cells, the nextsteps generally concern identifying the transformed cells for furtherculturing and plant regeneration. As mentioned herein, in order toimprove the ability to identify transformants, one may desire to employa selectable or screenable marker gene with a transformation vectorprepared in accordance with the invention. In this case, one would thengenerally assay the potentially transformed cell population by exposingthe cells to a selective agent or agents, or one would screen the cellsfor the desired marker gene trait.

(i) Selection

It is believed that DNA is introduced into only a small percentage oftarget cells in any one experiment. In order to provide an efficientsystem for identification of those cells receiving DNA and integratingit into their genomes one may employ a means for selecting those cellsthat are stably transformed. One exemplary embodiment of such a methodis to introduce into the host cell, a marker gene which confersresistance to some normally inhibitory agent, such as an antibiotic orherbicide. Examples of antibiotics which may be used include theaminoglycoside antibiotics neomycin, kanamycin and paromomycin, or theantibiotic hygromycin. Resistance to the aminoglycoside antibiotics isconferred by aminoglycoside phosphostransferase enzymes such as neomycinphosphotransferase II (NPT II) or NPT I, whereas resistance tohygromycin is conferred by hygromycin phosphotransferase.

Potentially transformed cells then are exposed to the selective agent.In the population of surviving cells will be those cells where,generally, the resistance-conferring gene has been integrated andexpressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of the exogenous DNA.

One herbicide which constitutes a desirable selection agent is the broadspectrum herbicide bialaphos. Bialaphos is a tripeptide antibioticproduced by Streptomyces hygroscopicus and is composed ofphosphinothricin (PPT), an analogue of L-glutamic acid, and twoL-alanine residues. Upon removal of the L-alanine residues byintracellular peptidases, the PPT is released and is a potent inhibitorof glutamine synthetase (GS), a pivotal enzyme involved in ammoniaassimilation and nitrogen metabolism (Ogawa et al., 1973). SyntheticPPT, the active ingredient in the herbicide Liberty™ also is effectiveas a selection agent. Inhibition of GS in plants by PPT causes the rapidaccumulation of ammonia and death of the plant cells.

The organism producing bialaphos and other species of the genusStreptomyces also synthesizes an enzyme phosphinothricin acetyltransferase (PAT) which is encoded by the bar gene in Streptomyceshygroscopicus and the pat gene in Streptomyces viridochromogenes. Theuse of the herbicide resistance gene encoding phosphinothricin acetyltransferase (PAT) is referred to in DE 3642 829 A, wherein the gene isisolated from Streptomyces viridochromogenes. In the bacterial sourceorganism, this enzyme acetylates the free amino group of PPT preventingauto-toxicity (Thompson et al., 1987). The bar gene has been cloned(Murakami et al., 1986; Thompson et al., 1987) and expressed intransgenic tobacco, tomato, potato (De Block et al., 1987) Brassica (DeBlock et al., 1989) and maize (U.S. Pat. No. 5,550,318). In previousreports, some transgenic plants which expressed the resistance gene werecompletely resistant to commercial formulations of PPT and bialaphos ingreenhouses.

Another example of a herbicide which is useful for selection oftransformed cell lines in the practice of the invention is the broadspectrum herbicide glyphosate. Glyphosate inhibits the action of theenzyme EPSPS which is active in the aromatic amino acid biosyntheticpathway. Inhibition of this enzyme leads to starvation for the aminoacids phenylalanine, tyrosine, and tryptophan and secondary metabolitesderived thereof U.S. Pat. No. 4,535,060 describes the isolation of EPSPSmutations which confer glyphosate resistance on the Salmonellatyphimurium gene for EPSPS, aroA. The EPSPS gene was cloned from Zeamays and mutations similar to those found in a glyphosate resistant aroAgene were introduced in vitro. Mutant genes encoding glyphosateresistant EPSPS enzymes are described in, for example, InternationalPatent WO 97/4103. The best characterized mutant EPSPS gene conferringglyphosate resistance comprises amino acid changes at residues 102 and106, although it is anticipated that other mutations will also be useful(PCT/WO97/4103).

To use the bar-bialaphos or the EPSPS-glyphosate selective system,bombarded tissue is cultured for 0-28 days on nonselective medium andsubsequently transferred to medium containing from 1-3 mg/l bialaphos or1-3 mM glyphosate as appropriate. While ranges of 1-3 mg/l bialaphos or1-3 mM glyphosate will typically be preferred, it is proposed thatranges of 0.1-50 mg/l bialaphos or 0.1-50 mM glyphosate will findutility in the practice of the invention. Tissue can be placed on anyporous, inert, solid or semi-solid support for bombardment, includingbut not limited to filters and solid culture medium. Bialaphos andglyphosate are provided as examples of agents suitable for selection oftransformants, but the technique of this invention is not limited tothem.

It further is contemplated that the herbicide DALAPON,2,2-dichloropropionic acid, may be useful for identification oftransformed cells. The enzyme 2,2-dichloropropionic acid dehalogenase(deh) inactivates the herbicidal activity of 2,2-dichloropropionic acidand therefore confers herbicidal resistance on cells or plantsexpressing a gene encoding the dehalogenase enzyme (Buchanan-Wollastonet al., 1992; U.S. patent application Ser. No. 08/113,561, filed Aug.25, 1993; U.S. Pat. No. 5,508,468; and U.S. Pat. No. 5,508,468; each ofthe disclosures of which is specifically incorporated herein byreference in its entirety).

Alternatively, a gene encoding anthranilate synthase, which confersresistance to certain amino acid analogs, e.g., 5-methyltryptophan or6-methyl anthranilate, may be useful as a selectable marker gene. Theuse of an anthranilate synthase gene as a selectable marker wasdescribed in U.S. Pat. No. 5,508,468; and U.S. patent application Ser.No. 08/604,789.

An example of a screenable marker trait is the red pigment producedunder the control of the R-locus in maize. This pigment may be detectedby culturing cells on a solid support containing nutrient media capableof supporting growth at this stage and selecting cells from colonies(visible aggregates of cells) that are pigmented. These cells may becultured further, either in suspension or on solid media. The R-locus isuseful for selection of transformants from bombarded immature embryos.In a similar fashion, the introduction of the C1 and B genes will resultin pigmented cells and/or tissues.

The enzyme luciferase may be used as a screenable marker in the contextof the present invention. In the presence of the substrate luciferin,cells expressing luciferase emit light which can be detected onphotographic or x-ray film, in a luminometer (or liquid scintillationcounter), by devices that enhance night vision, or by a highly lightsensitive video camera, such as a photon counting camera. All of theseassays are nondestructive and transformed cells may be cultured furtherfollowing identification. The photon counting camera is especiallyvaluable as it allows one to identify specific cells or groups of cellswhich are expressing luciferase and manipulate those in real time.Another screenable marker which may be used in a similar fashion is thegene coding for green fluorescent protein.

It further is contemplated that combinations of screenable andselectable markers will be useful for identification of transformedcells. In some cell or tissue types a selection agent, such as bialaphosor glyphosate, may either not provide enough killing activity to clearlyrecognize transformed cells or may cause substantial nonselectiveinhibition of transformants and nontransformants alike, thus causing theselection technique to not be effective. It is proposed that selectionwith a growth inhibiting compound, such as bialaphos or glyphosate atconcentrations below those that cause 100% inhibition followed byscreening of growing tissue for expression of a screenable marker genesuch as luciferase would allow one to recover transformants from cell ortissue types that are not amenable to selection alone. It is proposedthat combinations of selection and screening may enable one to identifytransformants in a wider variety of cell and tissue types. This may beefficiently achieved using a gene fusion between a selectable markergene and a screenable marker gene, for example, between an NPTII geneand a GFP gene.

(ii) Regeneration and Seed Production

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. In an exemplary embodiment, MS andN6 media may be modified by including further substances such as growthregulators. A preferred growth regulator for such purposes is dicamba or2,4-D. However, other growth regulators may be employed, including NAA,NAA+2,4-D or perhaps even picloram. Media improvement in these and likeways has been found to facilitate the growth of cells at specificdevelopmental stages. Tissue may be maintained on a basic media withgrowth regulators until sufficient tissue is available to begin plantregeneration efforts, or following repeated rounds of manual selection,until the morphology of the tissue is suitable for regeneration, atleast 2 wk, then transferred to media conducive to maturation ofembryoids. Cultures are transferred every 2 wk on this medium. Shootdevelopment will signal the time to transfer to medium lacking growthregulators.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, will then beallowed to mature into plants. Developing plantlets are transferred tosoiless plant growth mix, and hardened, e.g., in an environmentallycontrolled chamber at about 85% relative humidity, 600 ppm CO₂, and25-250 microeinsteins m⁻² s⁻¹ of light. Plants are preferably maturedeither in a growth chamber or greenhouse. Plants are regenerated fromabout 6 wk to 10 months after a transformant is identified, depending onthe initial tissue. During regeneration, cells are grown on solid mediain tissue culture vessels. Illustrative embodiments of such vessels arepetri dishes and Plant Cons. Regenerating plants are preferably grown atabout 19 to 28° C. After the regenerating plants have reached the stageof shoot and root development, they may be transferred to a greenhousefor further growth and testing.

Note, however, that seeds on transformed plants may occasionally requireembryo rescue due to cessation of seed development and prematuresenescence of plants. To rescue developing embryos, they are excisedfrom surface-disinfected seeds 10-20 days post-pollination and cultured.An embodiment of media used for culture at this stage comprises MSsalts, 2% sucrose, and 5.5 g/l agarose. In embryo rescue, large embryos(defined as greater than 3 mm in length) are germinated directly on anappropriate media. Embryos smaller than that may be cultured for 1 wk onmedia containing the above ingredients along with 10⁻⁵M abscisic acidand then transferred to growth regulator-free medium for germination.

Progeny may be recovered from transformed plants and tested forexpression of the exogenous expressible gene by localized application ofan appropriate substrate to plant parts such as leaves. In the case ofbar transformed plants, it was found that transformed parental plants(R₀) and their progeny of any generation tested exhibited nobialaphos-related necrosis after localized application of the herbicideBasta to leaves, if there was functional PAT activity in the plants asassessed by an in vitro enzymatic assay. All PAT positive progeny testedcontained bar, confirming that the presence of the enzyme and theresistance to bialaphos were associated with the transmission throughthe germline of the marker gene.

(iii) Characterization

To confirm the presence of the exogenous DNA or “transgene(s)” in theregenerating plants, a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays, such as Southernand Northern blotting and PCR™; “biochemical” assays, such as detectingthe presence of a protein product, e.g., by immunological means (ELISAsand Western blots) or by enzymatic function; plant part assays, such asleaf or root assays; and also, by analyzing the phenotype of the wholeregenerated plant.

1. DNA Integration, RNA Expression and Inheritance

Genomic DNA may be isolated from callus cell lines or any plant parts todetermine the presence of the exogenous gene through the use oftechniques well known to those skilled in the art. Note, that intactsequences will not always be present, presumably due to rearrangement ordeletion of sequences in the cell.

The presence of DNA elements introduced through the methods of thisinvention may be determined by polymerase chain reaction (PCR™). Usingthis technique discreet fragments of DNA are amplified and detected bygel electrophoresis. This type of analysis permits one to determinewhether a gene is present in a stable transformant, but does not proveintegration of the introduced gene into the host cell genome. It istypically the case, however, that DNA has been integrated into thegenome of all transformants that demonstrate the presence of the genethrough PCR™ analysis. In addition, it is not possible using PCR™techniques to determine whether transformants have exogenous genesintroduced into different sites in the genome, i.e., whethertransformants are of independent origin. It is contemplated that usingPCR™ techniques it would be possible to clone fragments of the hostgenomic DNA adjacent to an introduced gene.

Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition it is possible through Southernhybridization to demonstrate the presence of introduced genes in highmolecular weight DNA, i.e., confirm that the introduced gene has beenintegrated into the host cell genome. The technique of Southernhybridization provides information that is obtained using PCR™, e.g.,the presence of a gene, but also demonstrates integration into thegenome and characterizes each individual transformant.

It is contemplated that using the techniques of dot or slot blothybridization which are modifications of Southern hybridizationtechniques one could obtain the same information that is derived fromPCR™, e.g., the presence of a gene.

Both PCR™ and Southern hybridization techniques can be used todemonstrate transmission of a transgene to progeny. In most instancesthe characteristic Southern hybridization pattern for a giventransformant will segregate in progeny as one or more Mendelian genes(Spencer et al., 1992) indicating stable inheritance of the transgene.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA will only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR™ techniques also may be used for detection andquantitation of RNA produced from introduced genes. In this applicationof PCR™ it is first necessary to reverse transcribe RNA into DNA, usingenzymes such as reverse transcriptase, and then through the use ofconventional PCR™ techniques amplify the DNA. In most instances PCR™techniques, while useful, will not demonstrate integrity of the RNAproduct. Further information about the nature of the RNA product may beobtained by Northern blotting. This technique will demonstrate thepresence of an RNA species and give information about the integrity ofthat RNA. The presence or absence of an RNA species also can bedetermined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and will onlydemonstrate the presence or absence of an RNA species.

2. Gene Expression

While Southern blotting and PCR™ may be used to detect the gene(s) inquestion, they do not provide information as to whether thecorresponding protein is being expressed. Expression may be evaluated byspecifically identifying the protein products of the introduced genes orevaluating the phenotypic changes brought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

Assay procedures also may be used to identify the expression of proteinsby their functionality, especially the ability of enzymes to catalyzespecific chemical reactions involving specific substrates and products.These reactions may be followed by providing and quantifying the loss ofsubstrates or the generation of products of the reactions by physical orchemical procedures. Examples are as varied as the enzyme to be analyzedand may include assays for PAT enzymatic activity by followingproduction of radiolabeled acetylated phosphinothricin fromphosphinothricin and ¹⁴C-acetyl CoA or for anthranilate synthaseactivity by following loss of fluorescence of anthranilate, to name two.

Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of genesencoding enzymes or storage proteins which change amino acid compositionand may be detected by amino acid analysis, or by enzymes which changestarch quantity which may be analyzed by near infrared reflectancespectrometry. Morphological changes may include greater stature orthicker stalks. Most often changes in response of plants or plant partsto imposed treatments are evaluated under carefully controlledconditions termed bioassays.

IX. Assays of Transgene Expression

Assays may be employed with the instant invention for determination ofthe relative efficiency of transgene expression. Such methods would alsobe useful in evaluating, for example, random or site-specific mutants ofthe NOR sequences provided herein. Alternatively, assays could be usedto determine the efficacy of expression when various differentenhancers, terminators or other types of elements potentially used inthe preparation of transformation constructs.

For plants, expression assays may comprise a system utilizingembryogenic or non-embryogenic cells, or alternatively, whole plants. Anadvantage of using cellular assays is that regeneration of large numbersof plants is not required. However, the systems are limited in thatpromoter activity in the non-regenerated cells may not directlycorrelate with expression in a plant. Additionally, assays of tissue ordevelopmental specific promoters are generally not feasible.

The biological sample to be assayed may comprise nucleic acids isolatedfrom the cells of any plant material according to standard methodologies(Sambrook et al, 1989). The nucleic acid may be genomic DNA orfractionated or whole cell RNA. Where RNA is used, it may be desired toconvert the RNA to a complementary DNA. In one embodiment of theinvention, the RNA is whole cell RNA; in another, it is poly-A RNA.Normally, the nucleic acid is amplified.

Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g., ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology; Bellus, 1994).

Following detection, one may compare the results seen in a given plantwith a statistically significant reference group of non-transformedcontrol plants. Typically, the non-transformed control plants will be ofa genetic background similar to the transformed plants. In this way, itis possible to detect differences in the amount or kind of proteindetected in various transformed plants. Alternatively, clonal culturesof cells, for example, callus or an immature embryo, may be compared toother cells samples.

As indicated, a variety of different assays are contemplated in thescreening of cells or plants of the current invention and associatedpromoters. These techniques may in cases be used to detect for both thepresence and expression of the particular genes as well asrearrangements that may have occurred in the gene construct. Thetechniques include but are not limited to, fluorescent in situhybridization (FISH), direct DNA sequencing, pulsed field gelelectrophoresis (PFGE) analysis, Southern or Northern blotting,single-stranded conformation analysis (SSCA), RNAse protection assay,allele-specific oligonucleotide (ASO), dot blot analysis, denaturinggradient gel electrophoresis, RFLP and PCR™-SSCP.

(i) Quantitation of Gene Expression with Relative Quantitative RT-PCR™

Reverse transcription (RT) of RNA to cDNA followed by relativequantitative PCR™ (RT-PCR™) can be used to determine the relativeconcentrations of specific mRNA species isolated from plants. Bydetermining that the concentration of a specific mRNA species varies, itis shown that the gene encoding the specific mRNA species isdifferentially expressed. In this way, a promoters expression profilecan be rapidly identified, as can the efficacy with which the promoterdirects transgene expression.

In PCR™, the number of molecules of the amplified target DNA increase bya factor approaching two with every cycle of the reaction until somereagent becomes limiting. Thereafter, the rate of amplification becomesincreasingly diminished until there is no increase in the amplifiedtarget between cycles. If a graph is plotted in which the cycle numberis on the X axis and the log of the concentration of the amplifiedtarget DNA is on the Y axis, a curved line of characteristic shape isformed by connecting the plotted points. Beginning with the first cycle,the slope of the line is positive and constant. This is said to be thelinear portion of the curve. After a reagent becomes limiting, the slopeof the line begins to decrease and eventually becomes zero. At thispoint the concentration of the amplified target DNA becomes asymptoticto some fixed value. This is said to be the plateau portion of thecurve.

The concentration of the target DNA in the linear portion of the PCR™amplification is directly proportional to the starting concentration ofthe target before the reaction began. By determining the concentrationof the amplified products of the target DNA in PCR™ reactions that havecompleted the same number of cycles and are in their linear ranges, itis possible to determine the relative concentrations of the specifictarget sequence in the original DNA mixture. If the DNA mixtures arecDNAs synthesized from RNAs isolated from different tissues or cells,the relative abundances of the specific mRNA from which the targetsequence was derived can be determined for the respective tissues orcells. This direct proportionality between the concentration of the PCR™products and the relative mRNA abundances is only true in the linearrange of the PCR™ reaction.

The final concentration of the target DNA in the plateau portion of thecurve is determined by the availability of reagents in the reaction mixand is independent of the original concentration of target DNA.Therefore, the first condition that must be met before the relativeabundances of a mRNA species can be determined by RT-PCR™ for acollection of RNA populations is that the concentrations of theamplified PCR™ products must be sampled when the PCR™ reactions are inthe linear portion of their curves.

The second condition that must be met for an RT-PCR™ study tosuccessfully determine the relative abundances of a particular mRNAspecies is that relative concentrations of the amplifiable cDNAs must benormalized to some independent standard. The goal of an RT-PCR™ study isto determine the abundance of a particular mRNA species relative to theaverage abundance of all mRNA species in the sample.

Most protocols for competitive PCR™ utilize internal PCR™ standards thatare approximately as abundant as the target. These strategies areeffective if the products of the PCR™ amplifications are sampled duringtheir linear phases. If the products are sampled when the reactions areapproaching the plateau phase, then the less abundant product becomesrelatively over represented. Comparisons of relative abundances made formany different RNA samples, such as is the case when examining RNAsamples for differential expression, become distorted in such a way asto make differences in relative abundances of RNAs appear less than theyactually are. This is not a significant problem if the internal standardis much more abundant than the target. If the internal standard is moreabundant than the target, then direct linear comparisons can be madebetween RNA samples.

The above discussion describes theoretical considerations for an RT-PCR™assay for plant tissue. The problems inherent in plant tissue samplesare that they are of variable quantity (making normalizationproblematic), and that they are of variable quality (necessitating theco-amplification of a reliable internal control, preferably of largersize than the target). Both of these problems are overcome if theRT-PCR™ is performed as a relative quantitative RT-PCR™ with an internalstandard in which the internal standard is an amplifiable cDNA fragmentthat is larger than the target cDNA fragment and in which the abundanceof the mRNA encoding the internal standard is roughly 5-100 fold higherthan the mRNA encoding the target. This assay measures relativeabundance, not absolute abundance of the respective mRNA species.

Other studies may be performed using a more conventional relativequantitative RT-PCR™ assay with an external standard protocol. Theseassays sample the PCR™ products in the linear portion of theiramplification curves. The number of PCR™ cycles that are optimal forsampling must be empirically determined for each target cDNA fragment.In addition, the reverse transcriptase products of each RNA populationisolated from the various tissue samples must be carefully normalizedfor equal concentrations of amplifiable cDNAs. This consideration isvery important since the assay measures absolute mRNA abundance.Absolute mRNA abundance can be used as a measure of differential geneexpression only in normalized samples. While empirical determination ofthe linear range of the amplification curve and normalization of cDNApreparations are tedious and time consuming processes, the resultingRT-PCR™ assays can be superior to those derived from the relativequantitative RT-PCR™ assay with an internal standard.

One reason for this advantage is that without the internalstandard/competitor, all of the reagents can be converted into a singlePCR™ product in the linear range of the amplification curve, thusincreasing the sensitivity of the assay. Another reason is that withonly one PCR™ product, display of the product on an electrophoretic gelor another display method becomes less complex, has less background andis easier to interpret.

(ii) Marker Gene Expression

Markers represent an efficient means for assaying the expression oftransgenes. Using, for example, a selectable marker, one couldquantitatively determine the resistance conferred upon a plant or plantcell by a construct comprising the selectable marker coding regionoperably linked to the promoter to be assayed, e.g., an RS324 promoter.Alternatively, various plant parts could be exposed to a selective agentand the relative resistance provided in these parts quantified, therebyproviding an estimate of the tissue specific expression of the promoter.

Screenable markers constitute another efficient means for quantifyingthe expression of a given transgene. Potentially any screenable markercould be expressed and the marker gene product quantified, therebyproviding an estimate of the efficiency with which the promoter directsexpression of the transgene. Quantification can readily be carried outusing either visual means, or, for example, a photon counting device.

A preferred screenable marker gene assay for use with the currentinvention constitutes the use of the screenable marker geneβ-glucuronidase (GUS). Detection of GUS activity can be performedhistochemically using 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc) asthe substrate for the GUS enzyme, yielding a blue precipitate inside ofcells containing GUS activity. This assay has been described in detail(Jefferson, 1987). The blue coloration can then be visually scored, andestimates of expression efficiency thereby provided. GUS activity alsocan be determined by immunoblot analysis or a fluorometric GUS specificactivity assay (Jefferson, 1987).

(iii) Purification and Assays of Proteins

One means for determining the efficiency with which a particulartransgene is expressed is to purify and quantify a polypeptide expressedby the transgene. Protein purification techniques are well known tothose of skill in the art. These techniques involve, at one level, thecrude fractionation of the cellular milieu to polypeptide andnon-polypeptide fractions. Having separated the polypeptide from otherproteins, the polypeptide of interest may be further purified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography; polyacrylamidegel electrophoresis; and isoelectric focusing. A particularly efficientmethod of purifying peptides is fast protein liquid chromatography oreven HPLC.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide beingassayed always be provided in their most purified state. Indeed, it iscontemplated that less substantially purified products will have utilityin certain embodiments. Partial purification may be accomplished byusing fewer purification steps in combination, or by utilizing differentforms of the same general purification scheme. For example, it isappreciated that a cation-exchange column chromatography performedutilizing an HPLC apparatus will generally result in a greater “-fold”purification than the same technique utilizing a low pressurechromatography system. Methods exhibiting a lower degree of relativepurification may have advantages in total recovery of protein product,or in maintaining the activity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

A particular type of affinity chromatography useful in the purificationof carbohydrate containing compounds is lectin affinity chromatography.Lectins are a class of substances that bind to a variety ofpolysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclams and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

X. Oligonucleotide Probes and Primers

Naturally, the present invention also encompasses DNA segments that arecomplementary, or essentially complementary, to the sequences set forthin SEQ ID NO:1, SEQ ID NO:6, and SEQ ID NO:7. Nucleic acid sequencesthat are “complementary” are those that are capable of base-pairingaccording to the standard Watson-Crick complementary rules. As usedherein, the term “complementary sequences” means nucleic acid sequencesthat are substantially complementary, as may be assessed by the samenucleotide comparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:1, SEQ ID NO:6, orSEQ ID NO:7 under relatively stringent conditions such as thosedescribed herein. Such sequences may encode the entire NOR protein orfunctional or non-functional fragments thereof. Alternatively, thehybridizing segments may be shorter oligonucleotides. Sequences of 17bases long should occur only once in the genome of most plant speciesand, therefore, suffice to specify a unique target sequence. Althoughshorter oligomers are easier to make, numerous other factors areinvolved in determining the specificity of hybridization. Both bindingaffinity and sequence specificity of an oligonucleotide to itscomplementary target increases with increasing length. It iscontemplated that exemplary oligonucleotides of 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will beused, although others are contemplated. Longer polynucleotides encoding250, 500, 1000, 1212, 1500, 2000, 2500, or 3000 bases and longer arecontemplated as well. Such oligonucleotides will find use, for example,as probes in Southern and Northern blots and as primers in amplificationreactions.

Suitable hybridization conditions will be well known to those of skillin the art. In certain applications, for example, substitution of aminoacids by site-directed mutagenesis, it is appreciated that lowerstringency conditions are required. Under these conditions,hybridization may occur even though the sequences of probe and targetstrand are not perfectly complementary, but are mismatched at one ormore positions. Conditions may be rendered less stringent by increasingsalt concentration and decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Thus,hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

One method of using probes and primers of the present invention is inthe search for genes related to NOR from other species. Normally, thetarget DNA will be a genomic or cDNA library, although screening mayinvolve analysis of RNA molecules. By varying the stringency ofhybridization, and the region of the probe, different degrees ofhomology may be discovered.

Another way of exploiting probes and primers of the present invention isin site-directed, or site-specific mutagenesis. Site-specificmutagenesis is a technique useful in the preparation of individualpeptides, or biologically functional equivalent proteins or peptides,through specific mutagenesis of the underlying DNA. The techniquefurther provides a ready ability to prepare and test sequence variants,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

The technique typically employs a bacteriophage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, taking into account the degree ofmismatch when selecting hybridization conditions, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

XI. Breeding Plants of the Invention

In addition to direct transformation of a particular genotype with aconstruct prepared according to the current invention, transgenic plantsmay be made by crossing a plant having a construct of the invention to asecond plant lacking the construct. For example, a nucleic acid sequenceencoding a NOR coding sequence can be introduced into a particular plantvariety by crossing, without the need for ever directly transforming aplant of that given variety. Therefore, the current invention not onlyencompasses a plant directly created from cells which have beentransformed in accordance with the current invention, but also theprogeny of such plants. As used herein the term “progeny” denotes theoffspring of any generation of a parent plant prepared in accordancewith the instant invention, wherein the progeny comprises a constructprepared in accordance with the invention. “Crossing” a plant to providea plant line having one or more added transgenes relative to a startingplant line, as disclosed herein, is defined as the techniques thatresult in a transgene of the invention being introduced into a plantline by crossing a starting line with a donor plant line that comprisesa transgene of the invention. To achieve this one could, for example,perform the following steps:

-   -   (a) plant seeds of the first (starting line) and second (donor        plant line that comprises a transgene of the invention) parent        plants;    -   (b) grow the seeds of the first and second parent plants into        plants that bear flowers;    -   (c) pollinate a female flower of the first parent plant with the        pollen of the second parent plant; and    -   (d) harvest seeds produced on the parent plant bearing the        female flower.

Backcrossing is herein defined as the process including the steps of:

-   -   (a) crossing a plant of a first genotype containing a desired        gene, DNA sequence or element to a plant of a second genotype        lacking said desired gene, DNA sequence or element;    -   (b) selecting one or more progeny plant containing the desired        gene, DNA sequence or element;    -   (c) crossing the progeny plant to a plant of the second        genotype; and    -   (d) repeating steps (b) and (c) for the purpose of transferring        said desired gene, DNA sequence or element from a plant of a        first genotype to a plant of a second genotype.

Introgression of a DNA element into a plant genotype is defined as theresult of the process of backcross conversion. A plant genotype intowhich a DNA sequence has been introgressed may be referred to as abackcross converted genotype, line, inbred, or hybrid. Similarly a plantgenotype lacking said desired DNA sequence may be referred to as anunconverted genotype, line, inbred, or hybrid.

XII. Definitions

Genetic Transformation: A process of introducing a DNA sequence orconstruct (e.g., a vector or expression cassette) into a cell orprotoplast in which that exogenous DNA is incorporated into a chromosomeor is capable of autonomous replication.

Exogenous gene: A gene which is not normally present in a given hostgenome in the exogenous gene's present form In this respect, the geneitself may be native to the host genome, however, the exogenous genewill comprise the native gene altered by the addition or deletion of oneor more different regulatory elements.

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a coding DNA molecule such asa structural gene to produce a polypeptide.

Expression vector: A nucleic acid comprising one or more codingsequences which one desires to have expressed in a transgenic organism.

Progeny: Any subsequent generation, including the seeds and plantstherefrom, which is derived from a particular parental plant or set ofparental plants.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provide an expression control element for a structural gene and towhich RNA polymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

R₀ Transgenic Plant: A plant which has been directly transformed with aselected DNA or has been regenerated from a cell or cell cluster whichhas been transformed with a selected DNA.

Regeneration: The process of growing a plant from a plant cell (e.g.,plant protoplast, callus or explant).

Selected DNA: A DNA which one desires to have expressed in a transgenicplant, plant cell or plant part. A selected DNA may be native or foreignto a host genome, but where the selected DNA is present in the hostgenome, may include one or more regulatory or functional elements whichalter the expression profile of the selected gene relative to nativecopies of the gene.

Selected Gene: A gene which one desires to have expressed in atransgenic plant, plant cell or plant part. A selected gene may benative or foreign to a host genome, but where the selected gene ispresent in the host genome, will include one or more regulatory orfunctional elements which differ from native copies of the gene.

Transformation construct: A chimeric DNA molecule which is designed forintroduction into a host genome by genetic transformation. Preferredtransformation constructs will comprise all of the genetic elementsnecessary to direct the expression of one or more exogenous genes, forexample, NOR genes. Included within in this term are, for example,expression cassettes isolated from a starting vector molecule.

Transformed cell: A cell the DNA complement of which has been altered bythe introduction of an exogenous DNA molecule into that cell.

Transgene: A segment of DNA which has been incorporated into a hostgenome or is capable of autonomous replication in a host cell and iscapable of causing the expression of one or more cellular products.Exemplary transgenes will provide the host cell, or plants regeneratedtherefrom, with a novel phenotype relative to the correspondingnon-transformed cell or plant. Transgenes may be directly introducedinto a plant by genetic transformation, or may be inherited from a plantof any previous generation which was transformed with the DNA segment.

Transgenic plant: A plant or progeny plant of any subsequent generationderived therefrom, wherein the DNA of the plant or progeny thereofcontains an introduced exogenous DNA segment not originally present in anon-transgenic plant of the same strain. The transgenic plant mayadditionally contain sequences which are native to the plant beingtransformed, but wherein the “exogenous” gene has been altered in orderto alter the level or pattern of expression of the gene.

Transit peptide: A polypeptide sequence which is capable of directing apolypeptide to a particular organelle or other location within a cell.

XII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Genetic Mapping of Fruit Ripening Loci

Three F2 populations, each segregating for one of three ripeningmutants, were generated from interspecific crosses between L. esculentumcultivars homozygous for the respective mutant allele and a normallyripening L. cheesmannii parent (accession LA483). As a result RFLPmarkers less than one cM from both RIN (CT63-0.24 cM) and NOR (CT16-0.9cm) were identified.

A) RFLP Mapping of the rin Locus using Pooled DNA Samples: 1840 F2individuals segregating for the mutant rin allele and RFLP markers weregenerated from the interspecific cross between L. esculentum homozygousfor the mutant rin allele and L. cheesmannii homozygous for the alleleconferring the normal ripening phenotype. 45 DNA pools representing atotal of 225 mutant individuals (5 plants per pool) were generated fromthis population. Lanes 1-7 represented a subset of these pools and 35mutant individuals. Using this subset of DNA pools, the rin locus wasinitially mapped to a 10 cM region of chromosome 5 flanked by RFLPmarkers CT93 and TG448. Sample results from gel blot hybridization ofpooled DNA samples with RFLP probes are shown for TG448 (tightly linked)and TG185 (unlinked—greater than 50 cM away). “C” represents a DNA poolderived from 5 individuals with ripening fruit. “e” and “c” designatethe L. esculentum (mutant) and L. cheesmannii (normal) alleles of theRFLP probes employed, respectively. Hybridization to the “c” allele ofTG448 in pools 1, 3 and 6 represents individuals within these poolswhich have undergone recombination between the TG448 and rin loci. Theentire set of 45 pools was used to generate an RFLP map of the rinregion of chromosome 5.

B) Generation of linked markers via RAPD analysis of nearly isogenic DNApools: Methodology for isolating molecular markers linked to targetedregions of the genome is important in the event that markers closeenough for initiating a chromosome walk are not available. Untilrecently, methods for the isolation of molecular markers linked totargeted loci relied upon the availability of nearly isogenic lines(NILs) in which the target locus resides in a highly polymorphicintrogressed region (Young et al 1998, Martin et al 1991). Althoughnearly isogenic lines do exist for the rin, nor and Nr loci, in allcases, both the donor and recurrent parents used are L. esculentumcultivars. Unfortunately, extremely low levels of RFLP polymorphism aredetected among L. esculentum cultivars (Miller and Tanksley, 1990).Consequently, these NILs are not useful for the identification of newmarkers linked to ripening loci.

A new strategy was employed that rapidly identifies markers linked topreviously mapped target genes for which NILs are not available(Giovannoni et al 1991). This strategy is based on the generation ofnearly isogenic DNA pools from existing RFLP mapping populations. Inshort, once a target locus has been mapped to a genomic region betweenflanking RFLP markers, two DNA pools are generated from an RFLP mappingpopulation. Membership in either pool depends on the individuals'parental origin for the target region (as defined by scoring theflanking markers in the RFLP mapping population employed). The result istwo DNA pools representing individual progeny selected to be homozygousfor loci derived from either one parent or the other across the targetregion defined by the flanking markers. Since inclusion in a pool isdependent only on the parental origin of the target region, distantlylinked and unlinked loci are equally likely to be derived from eitherparent (i.e. they are not selected for). By combining DNA from multipleindividuals into each pool, the chances for homozygosity at loci otherthan those within the target region becomes minimal. The resulting DNApools are nearly isogenic for the targeted genomic region. These poolsare subsequently utilized as templates for RAPD reactions employingrandom primer PCR amplification of genomic templates (Williams, et al1990). Amplification products which differ between the two nearlyisogenic DNA pools are likely to be derived from the target region(Giovannoni et al, 1991).

In order to identify additional markers tightly linked to targetedripening loci, pairs of DNA pools nearly isogenic for all three ripeninglocus regions were generated and screened for polymorphic RAPD products.Random primers will be purchased from Operon Technologies and thepopulation used for the generation of isogenic DNA pools is the L.esculentum X L. pennellli F2 mapping population used to generate thetomato RFLP map. This population will be used rather than the actualripening gene mapping populations because of the higher degree ofpolymorphism between the parents (Miller and Tanksley, 1990). Thepurpose of this effort will be to identify tightly linked molecularprobes to be utilized in chromosome walks.

In previous analysis employing tomato genomic DNA templates, 3-7amplification products were detected, on average, per primer.Consequently, 500 primers should result in approximately 2,500 amplifiedloci of the tomato genome. Given the estimated map size of 1500 cM forthe tomato genome, 500 primers should yield approximately 1-2 markerswithin 1 cM on either side of a target locus. In the event thatsufficient polymorphic markers are not identified for one or more of thetarget loci, additional primers will be acquired through mutual primerexchanges. Also, the use of random primer pairs has recently beendemonstrated to yield significant numbers of unique amplificationproducts from tomato genomic DNA.

C) Isolation of a Molecular Marker Linked to the rin Locus usingIsogenic DNA Pools: Linkage analysis permitted the placement of the rinlocus between the markers TG503 and TG96. Progeny from an F2 population(L. esculentum X L. pennellii which were scored as homozygous for allmarkers between CT227 and TG318 were used to target the rin locus. DNAfrom 7-13 individuals was used to construct 2 nearly isogenic DNA pools.The region shown in black is the resulting chromosome target for theisolation of molecular markers linked to the rin locus. Numbers to theleft of the schematic chromosome designates recombination distances incM between markers designated on the right. A total of 100 random 10base primers were utilized for amplification of nearly isogenic DNApools resulting in one polymorphic PCR product, P76. The 0.5 kb P76amplification product was gel-purified, labeled and mapped via an EcoRIRFLP to the target region.

Example 2 Physical Mapping and Chromosome Walking to rin²

Following identification of DNA markers tightly linked to the RIN locus,physical mapping with high molecular weight DNA gel-blots was performedto assess the feasibility of initiating a chromosome walk. The resultwas identification of an 800 kb SmaI restriction fragment whichhybridized to 3 DNA markers which flanked the RIN locus and span agenetic distance of 4.2 cM. Based on this physical mapping data it wasestimated that one cM in the RIN region of chromosome 5 corresponds toapproximately 191 kb (800 kb/4.2 cM. =191 kb/cM). This estimate issimilar to that for the Pto locus which is linked to RIN (Martin et al.,1994).

Given the average 140 kb insert size of the tomato YAC library (Martinet al., 1992), a chromosome walk was initiated from the flanking singlecopy RFLP markers TG503, and CT93 which are 1.24 cM and 2.9 cM from rin,respectively. CT63, although only 0.24 cM from RIN on the TG503 side,was not employed as a probe because it is a member of a small genefamily and unlinked YAC clones would likely be recovered. However, a 360kb clone (Yrin2) demonstrated hybridization to both TG503 and CT63suggesting that 1) it contained sequences closer to the target locusthan any of the other clones, and 2) the estimated 191 kb/cM ratio inthe region of RIN was within two fold of accurate (i.e. TG503 and CT63are separated by 1 cM and reside on a 360 kb YAC). A single copy end ofYrin2 (Yrin2R) was isolated by inverse PCR and mapped, in a populationof 670 F2 progeny, on the RIN side of CT63 0.2 cM from rin. This end wassubsequently utilized to take a “step” toward RIN in the tomato YAClibrary resulting in the isolation of 4 additional YAC clones, Yrin8,Yrin9, Yrin11, and Yrin12. 7 of the 8 possible YAC ends were isolatedthrough inverse PCR and/or plasmid rescue for generation of a YAC contigvia cross-hybridization of ends with RIN region YACs, and RFLP mapping.Two YAC clones were determined to be chimeric based on RFLP mapping(Yrin9R) and sequencing of YAC ends (Yrin8R—greater than 95% homologywith tobacco chloroplast DNA). One YAC end, Yrin8L, cosegregated withRIN and hybridized to none of the other YAC clones, suggesting that itextended the furthest toward RIN and may harbor the target gene.

A) Completion of the chromosome walk to RIN: It was demonstrated that anend clone of a RIN region YAC designated Yrin8L cosegregates with RIN ina population of 670 F2 individuals. Based on high stringency (0.2×SSC,65° C.) DNA gel-blot analysis and RFLP mapping, Yrin8L, is a single copysequence in the tomato genome. Specific PCR primers were generated whichamplify the expected 270 bp fragment of Yrin8L from both the plasmidclone and the tomato genome. The primers were used to PCR screen thetomato YAC library of Martin et al. (1992). Screening of this YAClibrary via colony hybridization or PCR yielded 1-7 verified (viamapping of end clones and/or cross hybridization to 2 or more probesfrom the target region) recombinant clones for each of the 6 RIN or NORlinked markers tested. As an alternate strategy, random PstI, EcoRI, orHaeIII subclones of Yrin8 could be employed as probes in the next steptoward rin, and the random subclones tested for copy number, mapposition, and homology to Yrin YACs to ensure sequences from the end ofthe clone near RIN are used.

B) Isolation and characterization of cDNAs corresponding to genes withinYACs containing the target locus: Once candidate RIN containing YACclones were identified, the clones were used as probes for isolation ofcDNAs which may represent transcripts derived from genes contained onthe YAC. Two cDNAs were isolated and mapped using Ynor3 as a probe froma “Breaker” fruit cDNA, yielding numerous additional positive clones.Yrin8 also yielded numerous cDNA clones which were tightly linked to therin locus. A similar strategy was employed by Martin et al. (1993) inidentifying the tomato Pto gene on a 400 kb clone from this samelibrary.

cDNA libraries in the vector lambda gt10 and made from Mature Green,Breaker, and Red Ripe stage fruit are all available in the laboratory(lambda gt10 does not cross hybridize with the YAC vector pYAC4). All 3libraries will be screened because it is not known which stage willexpress the highest levels of target gene product. Construction of afourth cDNA library may also be used in lambda gt10 made from mRNAderived from several stages of immature fruit development. Specifically,mRNA will be combined from ovaries prior to pollination, 2 days postpollination, and every 10 days post-pollination up to the Mature Greenstage (approximately 30 days in cultivar Ailsa Craig). This library willhelp to minimize the problem of not knowing when during fruitdevelopment the RIN or NOR gene is expressed. Tissue for all but theunpollinated ovaries are stored in a −80° C. freezer.

Example 3 Physical Mapping and Chromosome Walking to NOR

High molecular weight DNA gel-blot analysis was also utilized toestimate the kb/cM ratio in the region of the NOR locus. A 1000 kb CspIfragment hybridized to both CT16 and TG313 which flank the NOR locus andare separated by approximately 5 cM. Based on this observation, it wasestimated that 1 cM in the region of the NOR locus corresponds toapproximately 200 kb.

Prior to initiation of the chromosome walk to nor, the closest knownflanking markers were CT16 (0.9 cM) and CT41 (2.3 cM). Based on thepublished tomato RFLP map (Tanksley et al., 1992), it was known thatTG395 resided in the interval between CT41 and CT16 and thus representeda closer marker to NOR than at least one of the two. TG395 was mapped asa sequence tagged site (due to lack of RFLPs between the two parents ofthe mapping population) to 1.4 cM from NOR on the CT41 side. PCRscreening of the tomato YAC library with TG395 specific primers resultedin the identification of 7 YAC clones ranging in size from 50 kb-490 kb.CT16 was not used for library screening because of difficulty ingenerating reliable PCR primers.

Three YAC ends were isolated via plasmid rescue and inverse PCR, asdescribed for RIN above. YAC end and RFLP marker cross hybridizationsyielded a YAC contig. Of particular interest was the observation thatthe 470 kb YAC, Ynor3, hybridized to both TG395 (which was used toretrieve this clone from the library) and CT16. Localization of NORwithin the interval of chromosome 10 bordered by TG395 and CT16suggested that the targeted NOR locus resides on Ynor3, and confirmedestimates of kb/cM in this region of the genome. A high titer cosmidlibrary (>500,000 clones) was then prepared of the yeast containingYnor3 to use in fine mapping and walking to NOR within the Ynor3 clone.The, cosmid vector employed, 04541, contains T-DNA borders to permitdirect Agrobacterium transfer into plants. Random Ynor3 PstI and HaeIIIsubclones were also isolated to use as fine mapping probes. A “breaker”stage tomato fruit cDNA library was also screened with Ynor3 as probe,yielding numerous clones for characterization for linkage to nor.

Example 4 Characterization of YAC Clones Harboring the rin and nor Loci

Initial screening with tightly linked RFLP markers yielded 6 YACs linkedto the nor locus and 5 YACs linked to rin. Hybridization to YACs withflanking markers revealed that a single nor YAC termed Ynor3 harboredthe target gene assuming no internal deletions or other perturbationswithin the YAC. Similar hybridization experiments, includinghybridization with isolated YAC ends, revealed that there were a numberof alterations in several of the YACs which presumably occurred duringlibrary construction and represented either deletions relative togenomic sequences or chimerism with fragments of genomic DNA derivedfrom unlinked regions of the tomato genome. Extensive characterizationof these clones was performed including RFLP mapping of random subclonesand YAC ends resulting in determination that the resulting YAC contigdid not extend to the point including the rin locus. The terminal YACend from the YAC extending closest to rin was subsequently used tore-screen the tomato YAC library resulting in identification of 4additional clones. One of these clones was extensively characterized dueto the presence (via hybridization) of DNA markers flanking rin on thissingle YAC. The results indicated that this particular clone harbored aninternal deletion resulting in the absence of the targeted rin locus.Following a third screen of the YAC library using a terminal YAC endfrom a previous screen as probe, a YAC termed Yrin11 was identifiedwhich harbored the rin locus as determined by random subcloning ofYrin11 restriction fragments and RFLP mapping to determine thatsequences flanking the target locus were contained on this YAC. This YACalso contained sequences that were absent from the YAC descried abovewith an internal deletion (thus confirming the integrity of Yrin11), andsaid fragments were tightly linked to the rin locus.

Example 5 cDNA Library Screening

A IX amplified breaker fruit cDNA library (cv. Ailsa Craig) in vectorlambda TRIPLEX was screened with whole PFGE gel-purified YACscorresponding to Ynor3 and Yrin11, respectively. A contig of tomato BACclones (library in pBELOBAC11, cultivar LA483-L. cheesmannii) was alsosimultaneously constructed across the nor region. Similar BAC contigefforts were initiated for the rin region of chromosome 5 but were notcompleted prior to isolation of the RIN gene. cDNAs hybridizing to thetwo candidate gene YACs, and thus potentially representing target genetranscripts, were verified for homology via hybridization as probes backto the respective Yrin11 and Ynor3 YACs. Positives were mapped as RFLPsand sequenced.

Example 6 NOR Gene Identification

Ynor3 hybridizing cDNA, CD11, was one of two clones found to cosegregatewith the nor locus and to hybridize to the BAC clone most likely (viahybridization to nor-linked markers) to harbor the target locus. BothCD11 and the other co-segregating cDNA (CD5) were hybridized to RNAgel-blots of normal and mutant fruit RNAs. CD5 was constitutivelyexpressed throughout fruit development while CD11 was induced duringripening and by ethylene. CD11 was also greatly reduced in expression infruit of the nor mutant. CD11 was sequenced and found to have homologyto the CUC (cup-shaped-cotyledon) gene of Arabidopsis. This gene alsohas homology to two functionally defined Arabidopsis transcriptionfactors of otherwise unknown function. Based on CD11 sequence, primerswere generated for RT-PCR of CD11 mRNA. CD11 alleles from normal(Nor/Nor) and mutant (nor/nor) tomato lines were generated by RT-PCR andsequenced. The mutant allele harbors a 2 bp deletion relative to thenormal CD11 allele resulting in a stop codon approximately mid-waythrough the CD11 open reading frame. Based on this mutation, and geneexpression patterns described above, in addition to the putative role ofCD11 as a transcription factor, it was indicated that the CD11 sequencerepresents the tomato NOR gene. An original sequence obtained of theclone is indicated in SEQ ID NO:1 and a corrected version of thesequence in SEQ ID NO:6.

Example 7 Confirmation of NOR Target Gene Isolation

The cDNA identified as representing NOR was be tested for function inripening through the use of antisense and sense expression constructs innormal and mutant tomatoes, respectively. Antisense gene suppression hasproven an effective tool for determining gene function during tomatofruit ripening (reviewed in Gray et al., 1994).

A) Preparation of NOR sense and antisense transformation constructs:T-DNA constructs were prepared for delivery of sense or antisense NORgene cDNA (CD-11) sequences into plant genomes (FIG. 1). First,NOR-pBI121 sense and antisense constructs were made by replacing the GUSgene of T-DNA binary vector pBI121 with the full-length NOR cDNAreferred to as CD-11 (1180 bp) in sense and antisense orientations,respectively, relative to the CaMV35S promoter (35S-P) of pBI121 (FIG.1). In both constructs, the NOR cDNA (CD-11 insert) was subclonedbetween SmaI and SacI restriction sites resulting from removal of theGUS gene from pBI121. Specifically, EcoRV and SacI sites of thepBluescript vector containing the NOR cDNA were employed due to the factthat SmaI and EcoRV (blunt) ends are compatible for ligation. Theresulting ligated sequence no longer can be digested with SmaI or EcoRV.Completed sense and antisense constructs were initially transformed intoE. coli DH 10B cells and then resulting plasmid DNA was isolated andtransformed into Agrobacterium tumefaciens strain LBA 4404 for use intransfer into the tomato gene as described herein.

The sense and antisense orientations of the NOR cDNA sequence relativeto the EcoRV and SacI restriction sites were obtained by subcloning theoriginal NOR cDNA bound by EcoRI sites from the original cDNA libraryvector (lambda gt10) into the EcoRI site of plasmid vector pBluescript.Due to the fact that the cDNA sequence was flanked by identicalrestriction sites (EcoRI), the insert could insert in either directionessentially at random. Several resulting NOR-pBluescript clones wereisolated and sequenced to determine the orientation of the cDNA insertrelative to the EcoRV and SacI sites of pBluescript, and one clonerepresenting each orientation (sense and antisense) was selected fortransfer into the SamI and SacI sites of pBI121 (following removal ofthe GUS gene from pBI121). In addition to the full-length NOR cDNA 3 bpof pBluescript polylinker (BS) was included on the SmaI side of the cDNAand 51 bp of pBluescript polylinker was included on the SacI side of theinsert (including the following restriction sites: SacII, NotI, Xbal,SpeI, BamHI, SmaI, PstI, EcoRI).

A sample of some of the classes of vectors that were prepared by theinventors for studies of the function of the NOR gene is given below, inTable 2. TABLE 2 Constructs for preparation of RIN and NOR transgenicplants: CONSTRUCT HOST PURPOSE 35s-antisense AC wild-type Phenocopy normutation CD5 35s-sense CD5 AC wild-type Ectopic expression of nor35s-sense CD5 MH1 nor/nor Complementation of nor mutant/nor confirmationGenomic CD5 MH1 nor/nor Complementation of nor mutant/nor confirmation

B) Transformation of wild type and mutant tomato plants: The sense andantisense NOR constructs prepared as described above were transformedinto wild type and nor mutant plants for confirmation of NOR identity. Amodified version of the transformation procedure described by Fillattiiet al. (1987) was used for generation of transgenic tomato plants(Deikman and Fischer, 1988).

Transgenic tomato plant were prepared as follows. First, the explant wasprepared by sterilizing seeds with soaking in 20% bleach +0.1% Tween-20for 15 minutes. The seeds were rinses 4 times in sterile distilled H₂Oand the seeds sown on MSO medium ((1 Liter): 4.0 g MS Salts (Gibco), 5.0ml B5 Vitamins, 5.0 ml MS Iron/EDTA, 20.0 g sucrose, 7.0 g agar(phytagar), pH medium to 6.0 with KOH, autoclave) in sterile glass jars,grow in growth chamber. Agrobacterium was then prepared by streakingselective fresh plates with Agrobacterium containing the desiredconstruct for 2-3 days before it was needed. A single colony was pickedfrom the plate and grown in tubes containing 2 ml YEP medium (YEP RichMedium (500 ml): 5.0 g Bacto-peptone, 2.5 g NaCl, 5.0 g Bacto YeastExtract, 7.5 g Bacto agar, autoclave) with appropriate antibiotics,followed by incubation on a shaker at 28° C. overnight. Explants wereprecultured two days before infection takes place, and 8-10 day oldcotyledons were excised. With sterile forceps, cotyledons were removedand placed onto an MSO plate. Using forceps and a blade, the cotyledonswere cut in 1-2 pieces. All pieces were then placed on pre-incubationmedium ((500 ml): 500 ml MSO Medium, 0.5 ml BAP, 0.1 ml IAA) in a deeppetri dish, wrapped in parafilm, and placed in growth chamber for 2days.

Overnight cultures of Agrobacterium were then precultured and spun downin a 4° C. centrifuge at 2800 rpm for 10 minutes, the supernatantdiscarded and pellets suspended in 10 ml of induction media ((100 ml): 5ml AB Salts, 2 ml MES buffer, 2 ml Sodium Phosphate buffer, 91 mldistilled water, 1 g glucose, autoclave, place in 10 tubes, 10 ml each).Then, for co-cultivation, the suspension was added to preculturedcotyledon pieces, wrapped in parafilm and shaken gently on roto-shakerfor 15 minutes. Using a spatula, cotyledon pieces were placed onco-cultivation media ((500 ml): 500 ml MSO Medium, 1 ml KH2PO4 (100mg/ml), 250 (l Kinetin (0.2 mg/ml), 100 (l 2,4 D (1 mg/ml), 735 (lacetosyringone)), wrapped in parafilm and placed in growth chamber for2-3 days.

For regeneration, after 2-3 days of being on co-cultivation medium,pieces were transferred to regeneration (2Z) medium ((500 ml): 500 mlMSO Medium, 5.0 ml Carbenicillin stock, 1.0 ml Kanamycin stock, 1.0 mlZeatin (1 mg/ml), 0.1 ml IAA), wrapped in parafilm and placed in agrowth chamber for 2-3 weeks. The tissue was transferred to fresh mediumevery 2-3 weeks. Generally, calli/shoots were apparent at 6 weeks. Thecalli was excised from cotyledon tissue and placed on fresh medium.Multiple shoots on one callus were separated and placed on medium,keeping all shoots together on one plate. The taller shoots were placedon deep petri dish. After shoots were well structured, anotherregeneration (1Z) medium (Regeneration Medium (1Z) (500 ml): 500 ml MSOMedium, 5.0 ml Carbenicillin stock, 1.0 ml Kanamycin stock, 0.5 mlZeatin (1 mg/ml), 0.1 ml IAA) could be utilized for conservingresources.

When shoots developed a well established meristem, individual shootswere excised of any remaining callus and placed in/on rooting medium((500 ml): 500 ml MSO medium, 1.0 ml Kanamycin stock, 0.2 ml IAA) inglass jars and placed in a growth chamber. The shoots were watched forsigns that: 1. callus continued, therefore suppressing roots to form; inthis instance the callus was cut off and again placed on fresh rootingmedia, or 2. if roots appeared, the plant was ready for soil.Transformed plantlets were then transferred from the glass jar andwashed off of any remaining agar on the roots under tap water gently andtransplanted in pot filled with moistened soil. The plantlets werewatered, making sure soil was thoroughly wet. Plantlets were coveredwith magenta box to conserve higher humidity. After 5-7 days, themagenta box was gradually removed. Plants were transferred to thegreenhouse grown to 10-15 cm.

Media used included the following: YEB Rich Medium (500 ml): 2.75 g BeefExtract, 0.55 g Yeast Extract, 2.75 g peptone, 2.75 g sucrose, 1 mlMgSO4 (1M) pH 7.2, 7.5 g Bactoagar, autoclave. B5 vitamins (100 ml): 2.0g myo-inositol, 0.2 g thiamine-HCl, 20.0 mg nicotinic acid, 20.0 mgpyridoxine-HCl, mix, then put in autoclaved bottle. MS Iron/EDTA (100ml): 556.0 mg FeSO4-7H₂O, 746.0 mg Na2EDTA-2H₂O, mix, then put inautoclaved bottle. Benzylamino-purine (BAP): 1 mg BAP/1 ml H₂O, dissolveBAP with 5N KOH dropwise, bring up to volume with ddH2O, filtersterilize in TC Hood, refrigerate. Zeatin: 1 mg Zeatin/1 ml H₂O,dissolve with 4N NaOH dropwise, bring up to volume with ddH₂O, filtersterilize in TC Hood, refrigerate. Indoleacetic acid (IAA): 1 mg IAA/1ml ethanol, dissolve with 100% ethanol, filter sterilize in TC Hood,refrigerate in foil (light sensitive, Good 1 week). Acetosyringone stock(ACE) (10 mg/ml): weigh out 100 mg of acetosyringone, add 10 ml 70%ethanol, filter sterilize, put in 1.5 ml tubes, place in −20C freezer.Carbenicillin Stock (50 mg/ml): weigh out 5 grams Carbenicillin, add 100ml ddH₂O, filter sterilize, put in 12 ml tubes, place in −20° C.freezer. Kanamycin Stock (50 mg/ml): weigh out 5 grams Kanamycin, add100 ml ddH₂O, filter sterilize, put in 12 ml tubes, place in −20Cfreezer. Tetracycline Stock (3 mg/ml): dissolve 3 mg tetracycline in 1ml ddH₂O, filter sterlize, refrigerate in foil (light sensitive), good 1day.

The results showed manipulation of fruit ripening and carotenoidaccumulation with the tomato NOR gene (FIG. 2). Shown in FIG. 2 arerepresentative control and transformed fruit from tomato a line of thegenotype nor/nor in the cultivar MH1 and transformed with NOR-pBI121Sense (FIG. 1). Primary transformants (T0) were confirmed for transgeneintegration via DNA gel-blot analysis and subsequently self-pollinated.Resulting seed were harvested and grown (T1 generation) and analyzed fortransgene segregation. Representative fully mature fruit from T1 nor/norindividuals that either harbor the sense NOR transgene (+) or havesegregated it out (−) are shown. In summary, transgene expression in themutant background was shown to partially recover the non-ripeningphenotype and confer ripening. In this particular line, relatively lowexpression of the transgene was observed as compared to expression ofNOR in normally ripening (Nor/Nor) fruit. Representative normal(Nor/Nor) and nearly isogenic mutant (nor/nor) cultivar MH1 tomato fruitare shown as controls. The partial recovery of ripening in the nor/norfruit harboring the NOR-pBI121 (+) transgene verified the isolation ofthe NOR gene. Furthermore, the partial ripening phenotype observed inthis line demonstrated that regulated expression of the NOR gene can beused to create a range of degrees of ripening and ripening-associatedcharacteristics (e.g., carotenoid accumulation, ripe flavor, nutrientcomposition, softness, pathogen susceptibility).

C) Considerations in complementation testing with genomic sequences:Several problems can arise when working with CaMV ³⁵S-cDNA constructsincluding 1) inappropriate level, developmental timing, or tissuespecificity of chimeric gene expression resulting in the absence of ameasurable phenotype in antisense or sense plants, and 2) induction ofgene expression in inappropriate cell types resulting in malformation orlethality in sense transformants. Because RIN represents a developmentalregulator whose activity could potentially prove deleterious tonon-fruit tissues, the ideal transgene would be under the control of thenormal RIN allele promoter, although other promoters with similarexpression profiles could provide similar advantages. Consequently, themajor emphasis in verification of putative cDNAs was placed oncomplementation of the mutant with corresponding genomic counterparts.

Genomic DNA sequences corresponding to the NOR cDNA were isolated fromthe tomato genomic library whose construction is described below (FIG.5). Full length cDNAs were sequenced at their termini, andoligonucleotide primers were be synthesized corresponding to the 5′ and3′ ends. Candidate genomic clones were then utilized as a template insequencing reactions with these end primers. Those genomic clonesharboring DNA sequences from both ends of the corresponding full lengthcDNA, as determined by sequencing, were restriction mapped to identifylocation of the transcribed region within the genomic clone insert.Restriction mapping, in combination with cDNA hybridization to genomicclone fragments, was utilized to identify genomic clones likely tocontain at least 2-3 kb of upstream and downstream sequence, prior totransformation. The sequence of the genomic DNA of the NOR generesulting from the analysis is given in FIG. 5.

D) Construction of target gene containing libraries in the cosmid/planttransformation vector: In order to facilitate generation of a contigspanning a target locus, libraries of genomic DNA from yeast containingYAC clones harboring the desired sequence were constructed using thecosmid/plant transformation vector 04541. The much smaller size of theyeast genome relative to tomato simplified the screening and contigconstruction. Libraries in 04541 were generated from yeast harboringYrin8 and Ynor3. Test screening of the Yrin8 library with CT63, Yrin2R,and Yrin8L demonstrated the presence of clones containing all threeprobed sequences. In addition, clones hybridizing to TG395, CT16, CDnor1and CDnor2 (the only 4 probes tested) were retrieved from the Ynor3library as well.

E) Walking in 04541 cosmid libraries from Yrin8L and CDnor2: DNA markersvery tightly linked to both RIN (Yrin8L) and NOR (CDnor2) wereidentified as described. No recombinations were identified between RINand Yrin8L in 670 F2 progeny, and only one recombinant between NOR andCDnor2 in 347 F2s. Based on the 200-300 kb/cM estimates for both the RINand NOR regions of the tomato genome, it was deemed reasonable toattempt a walk to both target loci from these linked markers as they arewithin the criteria set out for initiating development of a cosmidcontig. The walk from CDnor2 was initiated in the Ynor3 yeast cosmidlibrary, while that from Yrin8L was performed in the tomato genomiccosmid library described above because RIN may have been off the end ofYrin8. A DNA sequence surrounding the 04541 cloning site was generatedas were nested primers for IPCR of insert ends.

Example 8 Introgression of Transgenes Into Elite Crop Varieties

Backcrossing can be used to improve a starting plant. Backcrossingtransfers a specific desirable trait from one source to an inbred orother plant that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriategene(s) for the trait in question, for example, a construct prepared inaccordance with the current invention. The progeny of this cross firstare selected in the resultant progeny for the desired trait to betransferred from the non-recurrent parent, then the selected progeny aremated back to the superior recurrent parent (A). After five or morebackcross generations with selection for the desired trait, the progenyare hemizygous for loci controlling the characteristic beingtransferred, but are like the superior parent for most or almost allother genes. The last backcross generation would be selfed to giveprogeny which are pure breeding for the gene(s) being transferred, i.e.one or more transformation events.

Therefore, through a series a breeding manipulations, a selectedtransgene may be moved from one line into an entirely different linewithout the need for further recombinant manipulation. Transgenes arevaluable in that they typically behave genetically as any other gene andcan be manipulated by breeding techniques in a manner identical to anyother gene. Therefore, one may produce inbred plants which are truebreeding for one or more transgenes. By crossing different inbredplants, one may produce a large number of different hybrids withdifferent combinations of transgenes. In this way, plants may beproduced which have the desirable agronomic properties frequentlyassociated with hybrids (“hybrid vigor”), as well as the desirablecharacteristics imparted by one or more transgene(s).

Example 9 Marker Assisted Selection

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

In the process of marker assisted breeding, DNA sequences are used tofollow desirable agronomic traits (Tanksley et al., 1989) in the processof plant breeding. Marker assisted breeding may be undertaken asfollows. Seed of plants with the desired trait are planted in soil inthe greenhouse or in the field. Leaf tissue is harvested from the plantfor preparation of DNA at any point in growth at which approximately onegram of leaf tissue can be removed from the plant without compromisingthe viability of the plant. Genomic DNA is isolated using a proceduremodified from Shure et al. (1983). Approximately one gram of leaf tissuefrom a seedling is lypholyzed overnight in 15 ml polypropylene tubes.Freeze-dried tissue is ground to a powder in the tube using a glass rod.Powdered tissue is mixed thoroughly with 3 ml extraction buffer (7.0urea, 0.35 M NaCl, 0.05 M Tris-HCl pH 8.0, 0.01 M EDTA, 1% sarcosine).Tissue/buffer homogenate is extracted with 3 ml phenol/chloroform. Theaqueous phase is separated by centrifugation, and precipitated twiceusing 1/10 volume of 4.4 M ammonium acetate pH 5.2, and an equal volumeof isopropanol. The precipitate is washed with 75% ethanol andresuspended in 100-500 μl TE (0.01 M Tris-HCl, 0.001 M EDTA, pH 8.0).

Genomic DNA is then digested with a 3-fold excess of restrictionenzymes, electrophoresed through 0.8% agarose (FMC), and transferred(Southern, 1975) to Nytran (Schleicher and Schuell) using 10×SCP (20SCP: 2M NaCl, 0.6 M disodium phosphate, 0.02 M disodium EDTA). Thefilters are prehybridized in 6×SCP, 10% dextran sulfate, 2% sarcosine,and 500 μg/ml denatured salmon sperm DNA and ³²P-labeled probe generatedby random priming (Feinberg & Vogelstein, 1983). Hybridized filters arewashed in 2×SCP, 1% SDS at 650 for 30 minutes and visualized byautoradiography using Kodak XAR5 film. Genetic polymorphisms which aregenetically linked to traits of interest are thereby used to predict thepresence or absence of the traits of interest.

Those of skill in the art will recognize that there are many differentways to isolate DNA from plant tissues and that there are many differentprotocols for Southern hybridization that will produce identicalresults. Those of skill in the art will recognize that a Southern blotcan be stripped of radioactive probe following autoradiography andre-probed with a different probe. In this manner one may identify eachof the various transgenes that are present in the plant. Further, one ofskill in the art will recognize that any type of genetic marker which ispolymorphic at the region(s) of interest may be used for the purpose ofidentifying the relative presence or absence of a trait, and that suchinformation may be used for marker assisted breeding.

Each lane of a Southern blot represents DNA isolated from one plant.Through the use of multiplicity of gene integration events as probes onthe same genomic DNA blot, the integration event composition of eachplant may be determined. Correlations may be established between thecontributions of particular integration events to the phenotype of theplant. Only those plants that contain a desired combination ofintegration events may be advanced to maturity and used for pollination.DNA probes corresponding to particular transgene integration events areuseful markers during the course of plant breeding to identify andcombine particular integration events without having to grow the plantsand assay the plants for agronomic performance.

It is expected that one or more restriction enzymes will be used todigest genomic DNA, either singly or in combinations. One of skill inthe art will recognize that many different restriction enzymes will beuseful and the choice of restriction enzyme will depend on the DNAsequence of the transgene integration event that is used as a probe andthe DNA sequences in the genome surrounding the transgene. For a probe,one will want to use DNA or RNA sequences which will hybridize to theDNA used for transformation. One will select a restriction enzyme thatproduces a DNA fragment following hybridization that is identifiable asthe transgene integration event. Thus, particularly useful restrictionenzymes will be those which reveal polymorphisms that are geneticallylinked to specific transgenes or traits of interest.

Example 10 General Methods for Assays

DNA analysis of transformed plants is performed as follows. Genomic DNAis isolated using a procedure modified from Shure, et al., 1983.Approximately 1 gm callus or leaf tissue is ground to a fine powder inliquid nitrogen using a mortar and pestle. Powdered tissue is mixedthoroughly with 4 ml extraction buffer (7.0 M urea, 0.35 M NaCl, 0.05 MTris-HCl pH 8.0, 0.01 M EDTA, 1% sarcosine). Tissue/buffer homogenate isextracted with 4 ml phenol/chloroform. The aqueous phase is separated bycentrifugation, passed through Miracloth, and precipitated twice using1/10 volume of 4.4 M ammonium acetate, pH 5.2 and an equal volume ofisopropanol. The precipitate is washed with 70% ethanol and resuspendedin 200-500 μl TE (0.01 M Tris-HCl, 0.001 M EDTA, pH 8.0).

The presence of a DNA sequence in a transformed cell may be detectedthrough the use of polymerase chain reaction (PCR). Using this techniquespecific fragments of DNA can be amplified and detected followingagarose gel electrophoresis. For example, two hundred to 1000 ng genomicDNA is added to a reaction mix containing 10 mM Tris-HCl pH 8.3, 1.5 mMMgCl₂, 50 mM KCl, 0.1 mg/ml gelatin, 200 μM each dATP, dCTP, dGTP, dTTP,0.5 μM each forward and reverse DNA primers, 20% glycerol, and 2.5 unitsTaq DNA polymerase. The reaction is run in a thermal cycling machine asfollows: 3 minutes at 94° C., 39 repeats of the cycle 1 minute at 94°C., 1 minute at 50° C., 30 seconds at 72° C., followed by 5 minutes at72° C. Twenty μl of each reaction mix is run on a 3.5% NuSieve gel inTBE buffer (90 mM Tris-borate, 2 mM EDTA) at 50V for two to four hours.

For Southern blot analysis genomic DNA is digested with a 3-fold excessof restriction enzymes, electrophoresed through 0.8% agarose (FMC), andtransferred (Southern, 1975) to Nytran (Schleicher and Schuell) using10×SCP (20×SCP: 2 M NaCl, 0.6 M disodium phosphate, 0.02 M disodiumEDTA). Probes are labeled with ³²P using the random priming method(Boehringer Mannheim) and purified using Quik-Sep® spin columns (IsolabInc., Akron, Ohio). Filters are prehybridized at 65° C. in 6×SCP, 10%dextran sulfate, 2% sarcosine, and 500 μg/ml heparin (Chomet et al.,1987) for 15 min. Filters then are hybridized overnight at 65 C in 6×SCPcontaining 100 μg/ml denatured salmon sperm DNA and ³²P-labeled probe.Filters are washed in 2×SCP, 1% SDS at 65 C for 30 min. and visualizedby autoradiography using Kodak XAR5 film. For rehybridization, thefilters are boiled for 10 min. in distilled H₂O to remove the firstprobe and then prehybridized as described above.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe methods described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The references listed below are incorporated herein by reference to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

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1-64. (canceled)
 65. An isolated nucleic acid sequence comprising anantisense oligonucleotide complementary to a NOR gene mRNA encoded bySEQ ID NO:7.
 66. The isolated nucleic acid of claim 65, furthercomprising a promoter operably linked to said antisense oligonucleotide.67. The isolated nucleic acid of claim 65, further comprising anenhancer.
 68. The isolated nucleic acid of claim 67, wherein saidenhancer comprises an intron.
 69. The isolated nucleic acid of claim 65,comprising a transcriptional terminator.
 70. An expression vectorcomprising the isolated nucleic acid sequence of claim
 65. 71. Theexpression vector of claim 70, further defined as a linear nucleic acidsegment.
 72. The expression vector of claim 70, further defined as aplasmid vector.
 73. A plant transformed with the isolated nucleic acidsequence of claim
 65. 74. The transgenic plant of claim 73, furtherdefined as a fertile R₀ transgenic plant.
 75. The transgenic plant ofclaim 73, further defined as a progeny plant of any generation of afertile R₀ transgenic plant, wherein the progeny plant comprises theisolated nucleic acid sequence.
 76. A seed of the plant of claim 73,wherein the seed comprises the isolated nucleic acid sequence.
 77. Acell of the plant of claim
 73. 78. The plant of claim 73, furtherdefined as strawberry.
 79. A method of manipulating the fruit ripeningof a plant comprising introducing the expression vector of claim 70 intothe plant.
 80. The method of claim 79, wherein the expression vector isintroduced by a method comprising the steps of: (a) obtaining theexpression vector of claim 70; (b) transforming a recipient plant cellwith said expression vector; and (c) regenerating a transgenic plantfrom said recipient plant cell, wherein the fruit ripening phenotype ofsaid plant is altered based on the expression of the antisenseoligonucleotide.
 81. The method of claim 79, wherein the expressionvector is introduced by a method comprising the steps of: (a) obtaininga transgenic plant comprising the expression vector of claim 70; and (b)crossing the transgenic plant with itself or a second plant to introducethe expression vector into a progeny plant.
 82. The method of claim 79,wherein the plant is strawberry.